Development of New Tools for the Synthesis of "Difficult "

Marta Paradís Bas

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Tesi Doctoral

Development of New Tools for the

Synthesis of "Difficult Peptides"

Marta Paradís Bas

Dirigida i revisada per:

Dr. Fernando Albericio Dra. Judit Tulla Puche

(director i tutor) (directora)

(Universitat de Barcelona) (Institut de Recerca Biomèdica Barcelona)

Barcelona, 2015

Index

INDEX

Abbreviations and Acronyms i

ANNEXES

Annex I. Amino Acids iii

Annex II. Coupling Reagents and Additives iii

Annex III. Resins and Linkers iv

Annex IV. Protecting Groups iv

Overview of the thesis 1

GENERAL INTRODUCTION 5

1. "Difficult Peptides" and Aggregation 5

2. Strategies to Solubilize Peptides 10

OBJECTIVES 51

CHAPTER 1: Synthetic Approaches to Reach a "Difficult " 53

Introduction

1. The "Difficult Peptide" RADA-16 57

2. Solid-phase Fragment Condensation Approach 61

3. Fragment Condensation in Solution Approach 63

Publication I

RADA-16: A Tough Peptide–Strategies for Synthesis and Purification 71

Index

CHAPTER 2: A New Backbone Amide Protecting Group 85

Introduction

1. Safety-Catch Structure-Based 89

Publication II

2-Methoxy-4-methylsulfinylbenzyl: A Backbone Amide Safety-Catch Protecting Group for the Synthesis and Purification of Difficult Peptide Sequences 101

CHAPTER 3: Peptide-Based Solubilizing Tag Strategies 145

Introduction

1. Enhancement of Polarity by Peptide-Based Structures 149

2. C-Terminal Protecting Groups 152

Publication III

Linear versus Branched Poly-/ as Polarity Enhancer Tags 163

Publication IV

Semipermanent C-Terminal Carboxylic Acid Protecting Group: Application to Solubilizing Peptides and Fragment Condensation 179

GENERAL CONCLUSIONS 201

RESUM

Capítol 1 205

Capítol 2 210

Capítol 3 215

Abbreviations and Acronyms

Abbreviations and Acronyms

AA Ac acetyl ACH α-cyano-4-hydroxycinnamic acid ACN acetonitrile AM amino methyl Boc tert-butoxycarbonyl BTC bis(trichloromethyl) carbonate Bzl benzyl CD circular dichroism CM ChemMatrix CTC 2-chlorotrityl chloride d doublet DCHA dicyclohexylamine DCM dichloromethane DIEA N,N'-ethyldiisopropylamine DIPCDI N,N’-diisopropylcarbodiimide DMAP 4-(dimethylamino)pyridine DMF N,N'-dimethylformamide DMSO dimethyl sulfoxide EDT 1,2-ethanedithiol eq equivalents ESI electrospray ionization ESMS electrospray mass spectrometry FDA food and drug administration Fmoc 9-fluorenylmethoxycarbonyl HATU N-[(7-aza-1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N- methylmethanaminium hexafluorophosphate N-oxide HBTU N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N- methylmethanaminium hexafluorophosphate N-oxide HCTU N-[(6-chloro-1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N- methylmethanaminium hexafluorophosphate N-oxide HFIP 1,1,1,3,3,3-hexafluoro-2-propanol HOAt 1-hydroxy-7-azabenzotriazole HOBt 1-hydroxybenzotriazole HOSu N-hydroxysuccinimide HPLC high performance liquid chromatography HR-MS high resolution-mass spectrometry

i

Abbreviations and Acronyms

J coupling constant m multiplet MALDI-TOF matrix-assisted laser desorption/ionization-time-of-flight Mmsb 2-methoxy-4-methylsulfinylbenzyl Mmtb 2-methoxy-4-methylthiobenzyl MS mass spectrometry Mtt 4-methyltrityl MW microwave m/z mass-charge relation NMR nuclear magnetic resonance OSA 1-octane sulfonic acid OxymaPure 2-cyano-2-(hydroxyimino)acetate Pbf 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl PEG polyethylene glycol PG protecting group ppm parts per million PyBOP 1-benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate q quadruplet s singlet SPPS solid-phase t triplet TBTU N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N- methylmethanaminium tetrafluoroborate N-oxide tBu tert-butyl TFA trifluoroacetic acid TFE 2,2,2-trifluoroethanol TIS triisopropylsilane TMP 2,4,6-trimethylpyridine TMS tetramethylsilane tR retention time Trt trityl UV ultraviolet δ chemical shift

ii

Annexes

ANNEX I: Amino Acids

L- Ala A L-arginine Arg R L- Asp D L- Asn N L- Phe F

Gly G L- Glu E L- Gln Q L- His H L- Ile I

L- Leu L L-lysine Lys K L- Met M L- Pro P L- Ser S

L- Tyr Y L-triptophan Trp W L- Val V 1-naphthyl-L-alanine Nal

ANNEX II: Coupling Reagents and Additives

HOBt HBTU TBTU HCTU PyBOP

HOAt HATU OxymaPure HOSu DIPCDI

iii Annexes

ANNEX III: Resins and Linkers

CTC Resin Sieber Resin Rink Resin

(2-chlorotrityl chloride)

AM Resin HMPP Linker Mmsb-OH Linker

(aminomethyl) [4-(hydroxymethyl)phenoxy- (2-methoxy-4-methylsulfinyl- propionic acid] benzyl alcohol)

ANNEX IV: Protecting Groups

Trt Mtt tBu Pbf (trityl) (4-methyltrityl) (tert-butyl) (2,2,4,6,7-pentamethyl- dihydrobenzofuran-5-sulfonyl)

Mmsb Fmoc Boc (2-methoxy-4-methylsulfinylbenzyl) (9-fluorenylmethoxycarbonyl) (tert-butyloxycarbonyl)

iv Overview of the Thesis

Overview of the Thesis

The present thesis is structured as a compendium of publications which have been published in international scientific journals. These reports are organized in three chapters classified depending on the tool evaluated to achieve "difficult peptides" and aggregation prone peptides.

Initially, a General Introduction places the readers into the "difficult peptides" field and the peptide aggregation involvement during and after the synthesis. Furthermore, the strategies reported up to date address to overcome these synthetic and purification troubles are extensively described. The strategies comprised herein provide an overview about the two main branches of achieving these peptides by enhancing their solubility: during their synthesis or after their elongation. Moreover, the Objectives, according to the main subject of this thesis, address the aims proposed for each chapter.

Chapter 1 is focused on the known "difficult peptide" RADA-16. First, an Introduction is included in order to detail the general interest about this peptide and also to explain the basis of the synthetic strategies carried out in this project. Then, the specific research work addressed to obtain this peptide is detailed in the Results and Discussion and also the Experimental Part, both comprised in the Publication I section, as a full paper that includes also its supporting information.

Chapter 2 is based on the strategy which takes advantage of the use of backbone amide protecting groups to synthesize "difficult peptides". The Introduction provided is focused on certain concepts that have not been mentioned in the general introduction, but considered relevant to follow the experimental work. Then, in the Publication II section, experiments performed about this issue are described in a full paper with its supporting information as the Results and Discussion and also the Experimental Part of this chapter.

Chapter 3 contains two projects with the principal aim centered on facilitating the post-synthetic manipulation of non-soluble molecules by the short peptide-based solubilizing tag strategy. The two methodologies, the permanent and the temporary linkage have been performed in two differentiated projects. An additional proposal has been included in this chapter, which despite not being based on solubilizing strategies, takes the advantage of the same linker used for the temporary tag strategy. In this new approach, the linker has been explored as a carboxylic acid protecting group, including an application. An initial Introduction exposes the state of the strategies addressed to solubilize non-polar sequences by their conjugation to short peptide sequences as

1 Overview of the Thesis solubilizing tags, in order to expand specific details not included in the general introduction. Moreover, there are mentioned some of the most significant carboxylic acid C-terminal protecting groups described in the literature to provide an overview of the importance to contribute to this field. The Results and Discussion and also the Experimental Part of these projects are exposed in two independent papers, one letter and one communication, and both supporting information are included and presented in Publication III and Publication IV sections. Publication III, specifically covered a study about short peptide-based solubilizing tag strategies in permanent conjugations; and in the first part of publication IV, the same issue is addressed, although focused on temporary conjugations. The new application on carboxylic acid C-terminal protecting groups is found in the second part of the publication IV, where this group is applied in a fragment condensation synthesis.

Finally, the General Conclusions, organizes the main final accomplishments after the development of all the projects herein presented. In agreement with the objectives considered in the beginning, and according to the results obtained and presented in the publication sections, this last part shows the contribution of this thesis to the fields herein studied.

2

GENERAL INTRODUCTION

General Introduction

1. "Difficult Peptides" and Aggregation

The peptide synthesis has become an attainable process since 1963,1 when Professor Merrifield described the revolutionary concept of constructing these molecules by the solid-phase strategy. This invention was significant enough for him to win the Nobel Prize in Chemistry, at the same time that researchers received an extraordinary tool to facilitate the obtaining of peptides. After the solid-phase peptide synthesis (SPPS) was discovered, synthetic improvements have emerged addressed to the design of orthogonal protecting groups,2 new functionalized solid supports,3,4 efficient coupling reagents,5 or even methodologies to overcome difficulties on the sequence elongation. Nowadays, synthesis of large peptides has become achievable thanks to specific strategies emerged in recent years focusing on development of new synthetic tools. However, in spite of all the efforts invested in these fields, there are certain sequences still correlated with laborious syntheses.

The known as "difficult peptides" are an evident example of the need to continue developing strategies to enable their obtaining. This peptide concept was established in the ‘80s6 and refers to sequences that show inter- or intra-molecular β-sheet interactions significant enough to form aggregates. These structural associations occur during the peptide synthesis in both methodologies, solid-phase and solution-phase (Fig. 1)7 and they are stabilized and mediated by non-covalent hydrogen bonds, which, depending on the sequence, are favored. Specifically, they arise on the backbone of the peptide, in particular between the hydrogen amides and the carbonyls.8 The tendency of peptide chains to associate themselves is translated into a list of common behavioral features attributed to "difficult sequences", detailed by Kent9 and later by Milton.10 The main relevant synthetic evidences provided by these authors are the following: repetitive incomplete aminoacylations (<15%) despite re-couplings; accentuated difficulties when resin loading is high11 or when sterically hindered AAs are in the sequence; and slow or incomplete 9-fluorenylmethoxycarbonyl (Fmoc) removal.12

Parameters that N N favor aggregations :

H H Amino acid side-chain nature

Amino acid side-chain protecting group H H N α-substituent of amide bond N N Solvent type

Figure 1. Hydrogen bond inter-chain interactions during peptide synthesis (in solution or on solid-phase) and factors that contribute to their formation.

5 General Introduction

Some studies have analyzed the influence of each AA on β-sheet interactions, confirming that the capacity to induce intermolecular associations is sequence- dependent.13 Nevertheless, the prediction of a "difficult peptide" by the AAs present in its sequence, a priori, is not evident. Several factors that contribute to increase the propensity to form inter-chain associations are correlated with AA side-chains or even with the protecting group of the AA, with the nature of N-substitution of the backbone amide, and the characteristics of the solvent used to synthesize them (Fig. 1).10,14 Although the literature does not provide a comprehensive list of "difficult sequences", below we refer to some that fit the description and that have been synthesized, or their β-sheet folding mechanisms evaluated.

Sequences Peptide Type Ref. ionic RADA16-I [Ac-(RADARADA) -NH ] 46 4 2 self-assembling RADA16-II [Ac-(RARADADA) -NH ] 56 4 2 (Type I and Type II)

oligo-Ala, oligo-Val homooligo-pept. 17-23 oligo-Gln, oligo-Leu

Aβ (1-40) or Aβ (1-42) amyloidogenic 29-33

Amylin amyloidogenic 34, 35

ACP (65-74) 13, 20 - (H-VQAAIDYING-NH2) 57-60

HIV-1 PR (81-99) 20 - (H-PVNIIGRNLLTQIGCTLNF-NH2) 57-59

PnIA (A10) - 20, 59 (H-GCCSLPPCALNNPDYC-NH2)

Thymosin α1 - 61-64

Table 1. Common examples of sequences that aggregate on solid-phase: "difficult peptides".

On the other hand, there are peptides that form β-sheet associations in solution only once their sequences are fully unprotected, not during the elongation. These peptides assemble themselves in the liquid media establishing stable interactions that evolve to aggregates. Although that behavior may be considered an appreciated property in certain areas (explained in the following sections), in terms of peptide characterization or purification it supposes a significant drawback. These two post-synthetic steps demand sequences perfectly dissolved to provide an accurate peptide analysis. Again, this phenomenon is not easily predictable by the AAs present in the sequence, as it occurred for "difficult peptides". However, studies based on structural interaction experiments15 have led to develop computational methods to establish similarities between sequences that anticipate aggregation of peptides.16

6 General Introduction

Sequences Peptide Type Ref. Ac-V D-NH , Ac-A D-NH , 6 2 6 2 negatively charged Ac-V D -NH , Ac-L D -NH 40-42 6 2 2 6 2 2 surfactant-like Ac-GnD-OH (n=4,8,10) Ac-V K -NH , Ac-L K -NH , Ac-A K-NH , 6 2 2 6 2 2 6 2 positively charged Ac-KA -NH Ac-V H-NH , Ac-L K-NH , 41, 43 6 2, 6 2 6 2 surfactant-like H2V6-NH2, KV6-NH2 ionic KFE-8 [Ac-(FKFE) -NH ] 54 2 2 self-assembling KLD-12 [Ac-(KLDL)3-NH2] 46, 47 (Type I) ionic DAR16-IV [Ac-(A-(DA)4-(RA)3-R)-NH2] self-assembling 56 (Type IV) ionic ELK8-II (Ac-LELELKLK-NH2) self-assembling 56 (Type II) ionic EAH16-II [Ac-(AE) -(AH) -(EA) -(AH) -NH ] 2 2 2 2 2 self-assembling 56 EAK16-II [Ac-(EA) -(AK) -(AE) -(AK) -NH ] 2 2 2 2 2 (Type II) ionic RADSC-14 [Ac-(RADSC) -A -C-NH ] 2 5 2 self-assembling 55 RADSC-16 [Ac-(RADSC)3-A3-C-NH2] (Type I) Q7 (Ac-KFQFQFE-NH ) ionic 2 50-52 Q11 (Ac-QQKFQFQFEQQ-NH2) self-assembling

DN1 (Ac-QQRFQWQFEQQ-NH2) ionic 26 P11-4 (Ac-QQRFEWEFEQQ-NH ) self-assembling 43, 44 2

Sup35 (7-13) amyloidogenic 36, 37 (H-GNNQQNY-NH2)

Table 2. Common examples of sequences that aggregate in solution.

To sum up, aggregation in peptides can be originated in two different moments, during the synthesis or after the global deprotection of sequences. Herein, the peptides have been organized by families which present aggregation in one or both stages. Therefore, all kinds of peptides exposed below have in common the β-sheet association tendency, being highlighted if these interactions are presented in solution or during the synthesis (solid-phase).

1.1. Homooligo-Peptides

The homooligo-peptides are composed by only one AA type. These peptides are de novo synthetic sequences that show high tendency to aggregate, and are composed by hydrophobic AAs such as Ala, Gly, Ile, Leu or Val (Table 1). The aggregation is manifested not only during the synthesis of peptides on solid-phase, but also when the sequence is unprotected in solution. Those most commonly analyzed are poly•alanine and poly-valine,17 which show a notable capacity to assemble, in the case of oligo-alanine already starting from the fifth residue. Moreover, other homooligo sequences, such as poly(Gln) have also been explored regarding the aggregating mechanisms,18,19 or the poly(Leu) to validate new "difficult peptide" synthetic

7 General Introduction strategies.20 The driving force behind the assembly is the hydrogen bond formation between the backbone amides of the peptide chains. The authors who initially studied the behavior of these sequences, mentioned that, due to their characteristics, AA deletion occurred during the synthesis, thus demonstrating the structural conformation that explains this phenomenon.21–23 Especially, the oligo-alanine has been widely used as a model to study conformational changes after chemical modifications.

1.2. Self-Assembling Peptides

Self-assembling peptides (SAPs), as their name denotes, are another class of sequences characterized by their capacity to self-assemble. The self-assembling tendency appears when the peptide is completely unprotected after the cleavage from the resin, thus precluding its purification once in solution phase. These dissolved molecules spontaneously assemble into energetically favored β-structures that subsequently may form fibrils24 that may evolve to hydrogelation. The SAPs are not necessarily "difficult peptides", although some of them also may form β-sheet interactions during solid•phase synthesis and would also fit into this concept. Given their capacity to aggregate in solution, they provide a broad number of applications in biomedicine.25–27 Although self-assembly has been considered a desired property from a biomaterial perspective, this phenomenon underlies a number of human diseases.28 In this regard, probably the most known SAP is amyloid-β peptide (Aβ), the primary responsible agent of alzheimer’s disease.29,30 Specifically, the two amyloid sequences involved in this neurodegenerative illness are the 40-mer Aβ (1-40) and the 42-mer Aβ (1-42) (Table 1), which misfold and cause insoluble non-native disordered aggregates, forming oligomers and fibrils.31–33 Among the amyloidogenic peptides described, the (Table 1), another relevant sequence also present in humans, is highlighted because of its involvement in type 2 diabetes mellitus.34,35 This hormone is a 37-mer peptide secreted in , together with , and in a pathological situation, the excess of amylin self-assembles producing deposits in the pancreas and causing non- insulin dependent diabetes. One of the few amyloid-like peptides characterized by x- ray crystallography has been the 7-mer fragment of yeast prion Sup35, specifically the region (7-13)36 (Table 2). The Sup35 protein exists in a non-infectious form but when folded as an infectious variant is called a prion, in which the 7-mer region plays a key role. Moreover, studies on Sup35 heptapeptide fibril formation have been used to understand the aggregation mechanisms.37 Apart from the aforementioned peptides found in biological systems, there are other de novo designed sequences, classified as SAPs, which are not present in nature. These are commonly sub-divided into two families, described below.

8 General Introduction

1.2.1. Amphiphilic self-assembling peptides

The first family comprises amphiphilic (or amphipathic peptides, also named surfactant-like peptides),38,39 whose sequences have two differentiated parts, one hydrophobic (more than three non-polar AAs), and one hydrophilic (one or two polar or charged AAs). Depending on the length of the hydrophobic core, the solid-phase peptide synthesis may be hindered as occurs in "difficult peptides", being the largest chain the most difficult to achieve. Nevertheless, in solution all these amphiphilic self•assembling peptides tend to assemble by forming nanotubes or nanovesicles.40 Regarding their applications, it is worth highlighting the membrane protein stabilization allowed by some of these surfactant-like peptides.41 Sequences such as

V6D, A6D and V6K2 are examples of amphiphilic peptides which exhibit a demonstrated capacity to assemble, at certain concentration, in water solutions40–43 (Table 2). When the hydrophilic part of these peptides holds one kind of ionic charge, they are simply amphiphilic, whereas when they hold the two charges, they belong to the ionic peptide group.

1.2.2. Ionic self-assembling peptides

The sub-type of ionic self-assembling peptides are characterized by the stabilization of aggregates because of ionic interactions, being this effect caused by the positively and negatively charged AA side-chains.44,45 The ion-ion associations occurs once sequences are unprotected, thus the stabilization of aggregates in these peptides in solution is sever. Although most of the sequences belonging to this classification may be synthesized without difficulties on SPPS, there are some that also present β-sheet interactions during solid-phase synthesis, and therefore can also be classified in the group of "difficult peptides". Those de novo designs composed by rational arrangement of oppositely charged AAs, have served as self-assembling sequences (Table 1 and Table 2). These structures are sometimes named "peptide lego" and some of them exhibit supra-structures that have reached the market.46–48 Ionic self•assembling peptides are characterized mainly by the formation of complementary ionic interactions that are electrostatically orientated between two peptide chains facing opposite charges, thus favoring the self-assembling design when this phenomenon is desired. Peptide designs with alternating hydrophobic and hydrophilic AAs were among the initial methods proposed to achieve sequences with β-sheet interactions of interest.49–53 Later, Zhang, a relevant author in the field of well-defined self-assembling ionic sequences, proposed the division of these ionic self-assembling peptides into modules (I, II, III,...) (Table 1 and Table 2) on the basis of their positive and

9 General Introduction negative charge arrangement (type I: + − + − + −; type II: + + − − + +; type III: + + + − − − ; ... ).46,47,54–56

1.3. Other "Difficult Peptides"

Moreover, there are "difficult peptides" non-classified in a specific sub-type described in the literature. Some examples are the extensively used protein fragment named Acyl Carrier Protein [ACP (65-74)]13,20,57–60 (Table 1) or the fragment of human immunodeficiency virus protease [HIV-1 PR (81-99)]20,57–59 (Table 1), two models employed to evaluate new synthetic methodologies. Researchers specialized in conotoxin peptide synthesis have been used the Ala10→Leu mutant of PnIA [PnIA(A10L)] (Table 1) as a "difficult peptide" to validate synthetic methodologies,59 and it is also a sequence which has been used for the same proposal by other authors.20 Another example is the commercially available Thymalfasin (also known as α1)61 (Table 1) described for hepatitis B and C.62 It is a 28-mer peptide produced by the gland which participates in the mature of the T cells and with a wide range of reported medicinal applications,63 that have evolved to develop strategies to achieve it.64

2. Strategies to Solubilize Peptides

The main obstacle common to all "difficult peptides", and also to those sequences which aggregate in solution, is their insolubility. The non-soluble nature of peptides precludes their synthesis and/or hinders their characterization, and even prevents their purification. The peptide sequence and, most importantly, the AA composition, plays a key role in terms of secondary structure, thereby directly affecting the solubility of the molecules. The SPPS, and also the synthesis of peptides in solution, requires protected sequences to allow the two functional groups to react properly to lead the desired amide bond. The nature of these protecting groups contributes to enhance the hydrophobicity of the sequences and may induce interactions which favor the insolubility of peptides. In general, it is known that peptides that adopt α-helix secondary structures in solution are generally soluble in water, whereas β-sheet conformations are insoluble because of their capacity to interact by hydrophobic interactions which evolve to aggregates.

In order to overcome this drawback, several authors have invested efforts to develop strategies to enhance the solubility of peptides by disrupting the β-sheet interactions. Thus, the low solubility of some peptides is considered not only a barrier, but also a challenge in terms of developing new solubilizing methods. These methodologies can

10 General Introduction be classified in two principal groups, those that modulate an external factor and those that introduce a chemical modification to the peptide sequence.

2.1. External Factors

One of the first aspects to be considered when addressing peptide insolubility is the modification of external parameters. In some cases, external factors are introduced on solid-phase to favor peptide elongation by improving the AAs incorporation, or the protecting groups removal. These parameters may be introduced not only during the SPPS, but also to solubilize sequences in solution. Specifically, the main external parameters evaluated in the literature are the solvent, temperature, pH, the detergents and salts added to the peptide solution.

2.1.1. Solvent selection

Although N,N-dimethylformamide (DMF), a dipolar aprotic solvent, is commonly used in solid- and solution-phase peptide synthesis, several studies have explored the capacity of other solvents to substantially disrupt the secondary interactions that make peptides insoluble. In solid-phase the resin plays a key role in preventing intermolecular peptide associations. Consequently, an appropriate solvent to swell the resin may avoid these limitations, as well as the low loading may minimize the intermolecular connection.65 In some cases, dimethyl sulfoxide (DMSO) achieves solubilization of the hydrophobic sequences because, as a polar aprotic hydrogen bond-accepting solvent, it causes an increase in peptide mobility and consequently favors its solvation.66 A number of studies have demonstrated the use of DMSO as a solvent for SPPS to perform AA couplings and Fmoc removal treatments, thus enhancing the solubility and purity of the peptides.7 However, the oxidative properties of DMSO poses limitations for its use in SPPS and in solution when sequences contain AAs susceptible to oxidization, such as Met and Cys.

Another group of solvents that have been studied extensively and used to solubilize peptides in solution are the fluorinated alcohols, which the most widely used have been the 2,2,2•trifluoroethanol (TFE), the 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and the 1•phenyl•2,2,2-trifluoroethanol (PhTFE),67 (Fig. 2) among others. In contrast to non•fluorinated alcohols, the main characteristic of these fluorine-containing ones is their strong hydrogen-bond donor capacity, a property that makes them suitable for 67,68 interfering with the secondary structure of peptides. Mixtures of H2O/TFE in peptide copolymers with alternating neutral and charged AAs favors the α-helix when higher is the TFE proportion,69 supporting the general idea that TFE stabilizes the α-helix and

11 General Introduction only in some cases stabilizes β-helix.70 Furthermore, for certain peptide sequences there is a reversible α-β transition, that modulates the conformation of the peptide and consequently, its solubility.71,72 Thanks to the compatibility of fluorinated alcohols with solid-support protocols, HFIP has also been used to enhance solubility during the solid-phase synthesis of "difficult peptides", in those cases in which DMF is not as effective as a solvent. Its use has represented a suitable alternative for acylation, Fmoc removal,73 and even for peptide cyclization74. Other research groups have also explored the use of diverse solvents during SPPS to improve certain couplings.75,76 These solvents include tetrahydrofuran (THF), acetonitrile, and N-methyl-2-pirrolidone (NMP), which affect the swelling of the resin.65,77

2.1.2. Temperature increase

The peptide elongation on solid-phase, commonly performed at room temperature, may be carried out also at higher degree Celsius. Particularly, those synthesis performed at high temperatures are known as SPPS-elevated temperature (SPPS-ET). The most crucial aspects that must be considered when SPPS-ET is performed are the side-reactions, such as AA racemization, or the alteration of solvent-swelling on resin, among others. In the '90s, studies based on SPPS-ET developed protocols at 60 ºC with several coupling reagents, solvents or resins, which showed efficiency in peptide yields and purities.78 Furthermore, other reported peptide synthesis that followed this methodology demonstrated minimized racemization at several temperature ranges of 55-75 ºC,79 or 30-80 ºC80 for short peptides even using triethylamine. Moreover, similar protocols at 50 ºC may be used for large sequences for certain AA couplings, when under common conditions incorporations are hindered,65 or to facilitate "difficult peptide" synthesis.81 On the other hand, in solution peptide manipulation, the increase of temperature produces a disruption of their secondary structure that may enhance their solubility. This involvement on unfolding the peptide depends on the secondary structure transition of each self-assembling type sequence. Thus, modifying the characteristic β-sheet structure to a random coil by temperature is a described possibility to mediate the transition between insoluble into a soluble peptide.82

Another way to enhance the temperature in order to solubilize peptides relies on the microwave (MW)-assisted synthesis. Initial studies to perform combinatorial chemistry under MW conditions have been summarized years later,83 showing the large variety of organic reactions that can be accelerated thanks to these irradiations. Several reports have validated this strategy to enhance solubility which have allowed achieving the synthesis of a wide range of peptides.84–86

12 General Introduction

2.1.3. The pH control

When AAs in the peptide sequence are ionizable, positively or negatively, another external parameter to take into account is the pH, which allows modulation of the neat charge of the sequence in solution, thereby modifying its interactions with solvent and also its solubility.87,88 The pH value at which a sequence exhibits a conformational α-β transition depends on the pka of the AAs or on their isoelectric point. It is believed that the conformation of an ionizable peptide sequence depends more on external factors than on its intrinsic tendency to form a specific secondary structure. Several authors have taken advantage of ionizable peptides to control peptide interactions. In this regard, they have achieved interesting materials by modifying the pH.89–91 On SPPS, interfering in the pH of the system to enhance solubility is not common practice because this parameter directly affects the integrity of the protecting groups used in such synthesis and could lead to their removal or even cleavage of the peptide.

2.1.4. Salts addition

Other strategies to enhance the solubility of peptide sequences consist of adding chaotropic salts or denaturant agents (guanidinium chloride, urea or detergents) into the aqueous peptide solution. Regarding chaotropic salts, as their definition indicates, they are added to destabilize hydrophobic interactions and consequently the 92,93 aggregates. Lithium salts such as LiCl, LiBr or LiClO4 , are the most used for this purpose, together with other salts such as KSCN or NaClO4 (Fig. 2). These salts act by disrupting the intermolecular hydrogen-bonding, first increasing the polarity of the solvent (non-polar, aprotic or non-nucleophilic), and then displacing it by the most favorable ion-dipole interaction between the salt and the peptide. The associations of peptides in solution by ionic groups have been described by a number of researchers,94,95 and have stimulated other groups to address the addition of these salts to solubilize peptides, confirming it by NMR and circular dichroism. Those studies demonstrated that chaotropic salts can be used during the manipulation of a peptide in solution, and based on the same concept, during solid-phase synthesis to improve peptide synthesis, mainly when THF is used as solvent.96

13 General Introduction

LiCl δ LiI O - HFIP LiBF4 LiBr LiClO4 δ + KSCN S CaBr2

TFE MgBr2 DMSO ZnCl2 NaI

- + PhTFE DMF NMP

Figure 2. Common solvents and chaotropic agents used to enhance the solubility of peptides in solution and on solid-phase.

The addition, at a certain concentrations, of guanidinium hydrochloride (Gnd·HCl or Gdm·HCl) or urea to a peptide has resulted in increased solubility of the sequence, especially those containing non-polar AAs. The mechanism of action of these two agents suggests that there is a complex formation between the peptide backbone and the denaturant agent through hydrogen-bonds (2 hydrogen donors per agent; and 2 acceptors from carbonyl and amine).97 The stability of this interaction is stronger than the β-sheet association between peptide chains which form aggregation and this is translated into effective denaturant agents. Principally, guanidinium hydrochloride is used to dissolve peptides in solution or to prevent long peptide folding, by precluding the reaction, in peptide fragment ligations.98,99 NMR studies have confirmed these denaturant phenomenon based on the changes on chemical shift observed for structured conformations.100–102 Furthermore, given the effect of guanidinium hydrochloride on solubility, some studies have proposed the introduction of L-Arg in the aqueous solution of to improve their solubility.103,104 The extension of the arginine application to some non-soluble peptides was used to improve the peptide detection by HPLC. However, this is not an appropriate solubilizing method for the peptide synthesis on solid-phase neither in solution-phase, or even purification, as arginine addition may compete with AA coupling and it implies the presence of an extra impurity, which may hinder purification.

2.1.5. Detergent addition

Detergents are considered another kind of denaturing agent, and are also a useful additive for hydrophobic sequences. Although these agents are extensively used for proteins, the addition of some kinds has also been reported for peptides. The most commonly used detergents are the anionic and non-ionic sub-types, specifically sodium dodecyl sulphate (SDS) as an example of the former, and Triton X100 or Tween 20 as example of the latter (Table 3). They are used to solubilize peptides both in solution and during solid-phase synthesis, thus favoring yields and even peptide

14 General Introduction folding mechanisms.105 Detergents favor micelles formation, thus enhancing the aqueous solubility of peptides; these structural changes have been analyzed to further understand the solubilizing power of detergents.106,107 Some studies revealed that non•ionic detergents also preserve certain structured sequences, protecting the peptides in well-organized micelles.108,109 Although detergents have been commonly used for protein solubilization for many years,110,111 some lines of research recommend that they have to be avoided because may interfere with HPLC chromatography and decrease the signal intensity in mass spectrometry analysis.112 Recent publications have proposed protocols to improve HPLC compatibility with detergents.113–115 The most effective way to solubilize hydrophobic peptides or facilitate peptide reactions in solution is probably by combining appropriate solvents with certain detergents, the best known blend is the "magic mixture", which comprises dichloromethane (DCM)/DMF/NMP (1:1:1) containing 1% of Triton X-100 and 2 N of ethylene carbonate.116–120

Detergent Detergent Name Detergent Structure Type

Sodium dodecyl sulfate anionic (SDS)

Hexadecyltrimethyl ammonium bromide cationic

(CTAB)

Octyl non-ionic

glucopyranoside

Dodecyl maltopyranoside non-ionic

Big CHAP non-ionic

N-Decanoyl-N- methylglucamine non-ionic

(MEGA-10)

Genapol X-080 non-ionic

Table 3. Some detergents used in peptide synthesis to increase sequence solubility.

15 General Introduction

Detergent Detergent Name Detergent Structure Type

Triton X-100 non-ionic

X+Y+Z+W = 20

TWEEN 20 zwitter-ionic TWEEN 80 TWEEN 20 TWEEN 80

= R = R

CHAPS zwitter-ionic

Table 3. (Continued).

2.2. Chemical Modifications on the Peptide Sequence

In order to circumvent the limitations associated with some of the external factors, recent years have witnessed the development of other kinds of strategies. Specifically, more sophisticated tools to improve the solubility of peptides, focused on chemical modifications in the peptide sequences, have emerged. These structural changes modulate the physico-chemical properties of peptides. Chemical modifications introduced into the peptide sequence can be permanent as long as the newly introduced moieties do not affect the objective of study of the desired peptide. On the other hand, the incorporated chemical changes can be temporarily coupled to a certain functional group. In the latter case, peptides with enhanced solubility may be momentarily manipulated, and the solubilizing tool being removed later to afford the native peptide.

These strategies based on synthetic peptide modifications are comprised in two differentiated groups on the basis of the stage where the insolubility is originated: during the synthesis on solid-phase, or after the peptide cleavage, in solution. All these approaches are detailed below. Specifically, those tools designed to overcome the insolubility of peptides during solid-phase synthesis are addressed to synthesize "difficult peptides". On the other hand, those methods developed to dissolve sequences in solution are focused to facilitate the peptide characterization and purification.

16 General Introduction

2.2.1. Solubilization on solid-phase

2.2.1.1. Backbone amide protecting groups

The development of new protecting groups for peptide chemistry has focused mainly on α-amino, α-carboxylic acid, and on the side chain of functionalized AAs. However, orthogonal protection of backbone amides may be necessary in specific cases.2 In spite of the amides are functional groups commonly non-protected in peptide synthesis, there are a few number of relevant side-reactions where amides are involved in. The diketopiperazine (DKP)121,122 (Fig. 3b) or aspartimide123–125 (Fig. 3a) formation, both are initiated by the nitrogen from the amide bond under certain conditions.

a) i

basic or acid Nu H +

i ii ii

( Nu = , OMe, OH, NH2 ) b)

basic

Figure 3. Mechanisms involved in the formation of two side-products: (a) aspartimide; and (b) DKP.

The incorporation of a substituent in the amide position reduces the nucleophilicity of the nitrogen and subsequently precludes these internal cyclizations. Moreover, in addition to contribute to minimize these two by-products, this substitution on the amides becomes mandatory when the aggregation in SPPS needs to be avoided.

The backbone amide functions in peptides may be protected permanent- or temporarily. In the first type, the group remains on the sequence as a non-cleavable substituent (such as methyl groups); and in the second case, the group is named backbone amide protecting group, because it is removable under certain conditions. Numerous efforts have been addressed to design these kind of protecting groups, especially because they enable the synthesis of "difficult peptides".

2.2.1.1.1. Pseudoprolines

Pseudoprolines (ψpro), developed by Mutter in 1992,126,127 were the first backbone amide protecting groups described and supposed a crucial finding for the synthesis of "difficult peptides".128–130 These building blocks are based on the proline structure, as the name suggests. Previous reports, also by Mutter, proved the evident improvement

17 General Introduction of peptide synthesis when some AAs are substituted by .131–133 These benefits are due to the structural peculiarity of prolines, which has absence of the hydrogen on their α-amino group, which disrupts the continuity of hydrogen bonding on the backbone, a process responsible for the formation of insoluble aggregates. Furthermore, the 100% cis-amide conformation induced by the proline-like moiety destabilizes the β-sheet folding of peptides.133 Following these concepts, and in order to mimic the proline structure, Mutter built these derivatives based on thi/oxazolidines moieties. The are composed by any AA at the N-terminal position cycled by the hydroxyl or thiol groups from the side-chains of Ser, Thr, or Cys, which are placed in the C-terminal position (Fig. 4). The most common used pseudoprolines are those that contained dimethylation in the R1 position, the

Me,Me Me,Me dimethyloxazolidines AAx(ψ pro) and the dimethylthiazolidines AAx(ψ cys). In fact, pseudoprolines composed by almost all the AAs on its N-terminal are commercially available. The incorporation of pseudoprolines into a peptide sequence can be performed on solid-phase under standard coupling conditions. Once the peptide is cleaved from the resin by acydolysis, the pseudoproline is hydrolyzed, providing the two corresponding native AAs. Furthermore, many publications have demonstrated that this strategy greatly contributes to the synthesis of "difficult peptides",134 improves the cyclization of peptides,135 achieves large glycopeptides,136,137 and even facilitates protein synthesis.138,139 Despite the successful results attributed to the use of pseudoprolines in peptide sequences, their limitation resides in the necessity of having Ser, Thr or Cys in the sequence. This drawback has led to an increased demand to explore new backbone protecting groups.

ᴪpro cys Me,Me Fmoc-AA-AAx(ᴪ pro) SPPS (AA = Ser, Thr or Cys) x

cys X = O (Ser, Thr), S (Cys) TFA cleavage R2 = H (Ser, Cys), CH3 (Thr) R = any AA side-chain trans R1 = alkyl or phenyl

Figure 4. Pseudoproline structure and its incorporation in solid-phase peptide synthesis.

18 General Introduction

2.2.1.1.2. ortho-Hydroxybenzyl structure-based

Another research line focused on the design of backbone amide protectors was initiated by Sheppard and collaborators, also in the beginning of the ‘90s (1993), when they proposed the use of the N-(2-hydroxy-4-methoxybenzyl) (Hmb)14,140 moiety for Fmoc/tert-butyl (tBu) SPPS (see Fig. 5a and Table 4). The N-Fmoc-N(Hmb)-AAs is previously prepared in solution and then coupled on solid-phase to synthesize the desired peptide (Fig. 5a). After the Hmb-derived AA has been introduced into the sequence, the Fmoc group is removed, and the incoming AA is coupled through a non- standard mechanism (Fig. 5c). Thus, during the introduction of the incoming AA, the proton from the ortho-hydroxyl, in one of the tautomers, forms a hydrogen bond with the nitrogen of the secondary amine (α-amino) that favors the initial acylation at the hydroxyl position. Once this ester bond is formed, an intra-molecular O→N-acyl transfer occurs affording the desired amide bond and leaving the ortho-hydroxyl free. The peptide is elongated by standard SPPS conditions and, during the final peptide acidic cleavage from the resin, the Hmb is removed (see Table 4) from the sequence, together with other acid-labile protecting groups, affording the desired peptide target. a) Hmb derivative coupling

(X=coupling reagent) Hmb b) Phenol-PG c)

Fmoc

Ac O-acylation

Boc-Nmec

O →N acyl transfer Alloc

Figure 5. Hmb backbone protecting group: (a) mechanism during the coupling on peptidyl-resin with mono-N-Fmoc-N(Hmb)-AA derivative; (b) phenol protecting groups; and (c) mechanism of incoming amino acid incorporation on N(Hmb)-peptidyl-resin mediated by O→N acyl transfer.

Although some researchers have reported syntheses of peptides incorporating the building block as mono-N-Fmoc-N(Hmb) derivative,141,142 the major syntheses described, including the Sheppard proposal, have put forward the use of O,N•bisFmoc•N(Hmb) derivative (Fig. 5b).14,143,144 The main reason for synthesizing and

19 General Introduction incorporating Hmb-derivatives bearing extra Fmoc protection on the phenolic hydroxyl group is addressed to prevent the formation of the intermediate isolated and described by Nicolás and collaborators (see Fig. 5a).142 Thus, the mechanism that takes place during the incorporation of the mono-N-Fmoc-N(Hmb)AA occurs through an internal cyclization mediated by the nucleophilic attack of the hydroxyl to the activated carboxyl group. The product afforded is the lactone 4,5-dihydro-8-methoxy-1,4- benzoxazepin-2(3H)-one, which undergoes subsequent amino attack on the carbonyl to render the expected amide. This intra-molecular reaction is faster than the inter- molecular amino attack, and it has been demonstrated to be the main cause of the slow incorporation of Hmb-derivatives. In particular, the R substitution in lactone intermediate (see Fig. 5a) supposes a sterical hindrance when the amino group has to reach the carbonyl moiety. That sterical obstacle is the principal cause of the poor reactivity of lactone and consequently slow incorporation of Hmb-derivative. This drawback can be solved with the protection of the phenolic hydroxyl group (Fig. 5b).

When Fmoc is used to protect the ortho-hydroxyl from Hmb, the protection is temporary. Commonly, once the Fmoc group is removed in a standard peptide synthesis, the hydroxyl is left free, which does not affect subsequent AA incorporations. However, when certain molecules susceptible to react with this hydroxyl group are incorporated into the sequence, the semipermanent protection of phenol group becomes mandatory, being possible to protect it directly on resin (Fig. 5b). One strategy usually chosen is the acetylation (AcHmb), which is performed on solid-phase by acetic anhydride in presence of DIEA. The AcHmb removal occurred in two steps, first, the hydrazine treatment to afford the Hmb and second, the TFA cleavage conditions to release the unprotected peptide.145 Some authors have adopted the option of acetylation145–148 while others have selected alternatives to protect the phenol from Hmb, such as the use of the allyloxycarbonyl group (Alloc)145 or the tert- butoxycarbonyl•N'•methyl-N-[2-(methylamino)ethyl]carbamoyl (BocNmec)149 (Fig. 5b). In a similar process as acetylation, Alloc is introduced on solid-phase by diallyl pyrocarbonate (Alloc2O) in presence of DIEA; and the BocNmecHmb is incorporated by activating the phenol with p-nitro-phenylchloroformate followed by a treatment with mono-Boc-N,N-dimethylethylenamine/N,N'-ethyldiisopropylamine (DIEA) in DCM.

Removal of AllocHmb and NmecHmb required also two steps, first, the Pd(PPh3)4 treatment (for Alloc)150 or the N-methylmorpholine (for Nmec);151 and second, the standard TFA Hmb removal mentioned before.

On the other hand, the ester bond formation when the AA is being coupled onto the N(Hmb)-peptidyl-resin occurs kinetically slower than an standard amide bond, and this

20 General Introduction is translated into a prolonged final O→N-acyl shift. In order to avoid possible side•reactions derived from this slow acylation, it was proposed that the dipeptide

Fmoc-AA2-N(Hmb)-AA1-OH would be pre-synthesized in solution and further introduced into the sequence. Although aspartimide prevention has been reported not only for synthesis which used mono-N-Fmoc-N(Hmb)-AA,152 but also when using the 153 dipeptide Fmoc-AA2-N(Hmb)-AA1-OH, slow incorporations of the bulky dipeptide leading to racemization153 have been observed.

In spite of these limitations attributed to Hmb backbone amide protector, some dipeptides containing this protecting group are commercially available. Over the years, a number of peptide syntheses81,152,154,155 have described that Hmb inhibits aspartimide formation and enhances the solubility of peptides facilitating the synthesis of "difficult peptides". Specifically, aggregations are abolished in a peptide when Hmb is introduced into the sequence after the fifth or sixth residue.

Parallel to the use of Hmb for the Fmoc/tBu strategy, an equivalent backbone amide protecting group suitable for Boc/benzyl (Bzl) SPPS strategy was required in order to cover the principal peptide strategies on solid-phase. In this regard, in 1994, two of Sheppard’s collaborators for the Hmb proposal, Johnson and Quibell, defined the N•(2•hydroxybenzyl) group (Hbz)156 (Table 4). This group shows enhanced acid stability compared with Hmb and is therefore resistant to the continuous TFA treatments required to remove the temporary Boc groups common in Boc/Bzl SPPS strategies. The authors described the preparation of a modified AA protected by Hbz in solution and analogous to the Hmb derivatives, obtaining the O,N-bisFmoc-N(Hbz)-AA derivative. Although the synthesis was performed using the Fmoc/tBu strategy, they demonstrated the stability of the protecting group to TFA and its final removal by a mixture of trifluoromethanesulfonic acid and TFA (Table 4). The coupling of the incoming AA onto the N(Hbz)-peptidyl-resin occurs more slowly than in Hmb-containing peptidyl-resin, probably because of the absence of the electron-withdrawing effect of the methoxy in meta position respect the hydroxyl group.

Other approaches to prevent the poor O→N-acyl transfer associated with Hmb or Hbz have relied on the development of new backbone protecting groups that modify the benzyl substituent to afford a more efficient transacylation. In 1999, Meutermans and Smythe described a new nitro-activated group, the 6-nitro-2-hydroxybenzyl (Hnb)157–159

(Table 4). The electron-withdrawing nitro group decreases the pKa of the ortho ionizable group, thus favoring O-acylation and enhancing the reactivity of the O→N•acyl transfer. Incorporation of this group was performed in a different manner to that used for the ortho-hydroxybenzyl analogs described previously. In this case,

21 General Introduction

6•nitro-2-hydroxybenzaldehyde is reacted with the α-amino from the peptidyl-resin by a reductive amination. The peculiarity of nitro derivatives is the process by which they are removed, which is mediated by photolysis at a certain wavelength leaving the nitrosobenzaldehyde.160

1,3-Benzoxathiole- Hmb Hmsb 3-oxide derivative Hbz Hnb

TFA/phenol/EDT/TES TFA/TMSBr/EDT/thioanisole λ= 366 nm UV light NH I/Me S in TFA (92:3:3:2 v/v), 2 h, rt (89:4.5:2:4.5 v/v), 2 h, rt 4 2 TFA/EDT/thioanisole/TFMSA (1 mM in or or (20:20 eq) 1% AcOH/MeOH at 1mg/mL, 2 h, 0 ºC (78:4:8:10 v/v), 2 h, rt

TFA/phenol/TIS SiCl4/TFA/EDT/anisole or 1% AcOH/DMSO REMOVAL

CONDITIONS (94:3:3 v/v), 2 h, rt (5.:90:2.5:2.5 v/v), 2 h, rt 2-3 h, rt

14, 81, 140-149, 20, 162, 163 161 156 157-159 REF. 152-155

Table 4. Backbone amide protecting groups found in the literature based on o-hydroxybenzyl structure.

Subsequent to the Hmb group, and based on the ortho-hydroxy moiety which enables the O-N rearrangement, in 1997, Offer together with Quibell and Johnson, proposed the introduction of an electron-withdrawing group in para position to the 2-hydroxyl function, specifically the benzoaxazepsin-2(3H)-one moiety. This group, the (6-hydroxy- 3-oxido-1,3-benz[d]oxathiol-5-yl)methyl (1,3-Benzoxathiole-3-oxide derivative),161 (Table 4) can be used both in Boc and Fmoc strategies and the advantages associated with the modification on the Hmb protector are similar to those offered by nitro derivatives, since both groups share the same chemical electron properties. These authors proved that acylation occurs at a higher rate in less potent coupling conditions, thus considerably suppressing the epimerization side-reaction promoted by a long acylation. Moreover, the cleavage of the protecting group is completed after a reductive-acidolysis treatment (although it was not designed for this proposal, it may be considered a "safety-catch" protecting group, concept explained in the introduction of chapter 2). Along these lines, some years later, Quibell and Johnson presented the N-(3-methylsulfinyl-4-methoxy-6-hydroxybenzyl (SiMB, also named Hmsb)162 (Table 4), a new generation of "safety-catch" protecting groups (see chapter 2) that was easier to synthesize and showed equivalent acylation kinetics to the previous one. Quibell and collaborators have recently described new applications for the Hmsb group that support the efficiency of synthesizing "difficult peptides" when this protecting group is used.163 More recently, one research group has also focused their attention on the Hmsb amide backbone protection, and have studied specifically the optimization of coupling conditions, as well as its application in some difficult large sequences.20

22 General Introduction

Furthermore, the Hmsb group provides an additional advantage attributed to the fact that since it is acid resistant, it gives the possibility of obtaining the Hmsb-containing sequences after TFA cleavages. This property makes it a "safety•catch" protecting group, a concept that has been further studied in the introduction of chapter 2.

2.2.1.1.3. ortho-Mercaptobenzyl structure-based

Regarding the same O→N-acyl transfer mechanism associated to Hmb-based protectors to favor the amide bond formation, several researchers described similar protecting groups, but addressed to a different proposal, specifically to assist the obtaining of large peptides/proteins through the of unprotected peptide fragments in solution164 (Fig. 6). Among all the described auxiliaries for the N•terminus, herein, two of them based on a mercaptobenzyl scaffold have been highlighted. In spite of not being designed as a solubilizing tool, we have considered these mercaptobenzyl derivatives as backbone protecting groups because, after the fragment ligation, these groups become amide bond protectors.

B B A A + exchange transfer

A B A B cleavage

Figure 6. Auxiliary-mediated peptide fragment ligation. Peptides A and B are unprotected sequences and R can be a hydrogen or a methoxy group.

In the earlier 2000s, Dawson165,166 and Aimoto,167 in parallel, developed the mercaptobenzyl moiety as an N-terminal protecting group. In particular, they defined two analogues, the di-methoxy derivative 4,5-dimethoxy-2-mercaptobenzyl (Dmmb,166,167 also named Dmb, abbreviation that unfortunately was also selected for another protector, see 2.2.1.1.4. section) (Table 5); and the tri-substituted 4,5,6•trimethoxy-2-mercaptobenzyl (Tmb,166 abbreviation that unfortunately was also selected for another protector, see 2.2.1.1.4. section) (Table 5).

The incorporation of the Dmmb group on the peptidyl-resin can be performed by two different methods based on a reductive amination mechanism. In one case, the reaction occurs between the amino group contained in the mercaptobenzyl moiety167 and the aldehyde present on the N-terminal peptidyl-resin. In another case, the incorporation involves an aldehyde group contained in the mercaptobenzyl moiety165

23 General Introduction and an amino group present on the N-terminal peptidyl-resin. On the other hand, the Tmb group is introduced into the sequence by reacting the amino group contained in the mercaptobenzyl moiety through a nucleophilic attack on an acid bromide 166 N•terminal peptidyl-resin.

Dmmb Tmb

1 M TFMSA/1 M thioanisole in TFA TFA/TIS

1 h, 0 ºC (95:5 v/v), 2 h, rt

REMOVAL CONDITIONS

166, 167 166 REF.

Table 5. Backbone amide protecting groups found in the literature based on o-mercaptobenzyl structure.

The incoming AA is coupled onto the N(Hmb)-peptidyl-resin through an S→N-acyl transfer, which occurs in an equivalent manner as the O→N-acyl shift in o-hydroxybenzyl-based protectors. Dawson and collaborators demonstrated that for chemical ligation, the Dmmb group allows the acylation of the incoming AA faster than when Hmb derivative is used.166 The final Dmmb and Tmb removal, (Table 5) which affords the desired peptide target, is carried out during the peptide acidolytic cleavage from the resin, together with other acid-labile protecting groups. It is worth highlighting that Dmmb removal is slower than that for Tmb, the former requiring strong acids, such as TFMSA or HF.

2.2.1.1.4. para-Methoxybenzyl structure-based

Several backbone amide protectors that are not based on the ortho-hydroxyl moiety, are composed by methoxy substituted benzyl structures addressed to enhance the TFA lability to remove them during cleavage of the peptide. The first of those groups proposed was the 2,4-dimethoxybenzyl (Dmob, also named Dmb),168 defined initially as backbone amide protecting group for solution-phase synthesis in 1966 (Table 6). The ortho and para electron-donating substitution of these protectors allows their fast removal at high concentrations of TFA. It is described that the proton of the amino group forms an internal hydrogen bond with the oxygen from the o-methoxy function, equivalently to the o-hydroxy moiety in Hmb derivative (see Fig. 5c), which favors the acylation. Nevertheless, the bulkiness of the methoxy group induces severe steric hindrance that, in some cases, may preclude the incorporation of the following AA. In

24 General Introduction spite of this limitation, Zahariev and White, independently, demonstrated the efficiency of Dmb in preventing aspartimide side-reactions.169,170

Dmb Tmob 4-methoxy-2-Nb

366 nm UV light TFA/scavengers TFA/scavengers (in MeCN/H2O+ 0.1% TFA (pH 2) (95:5 v/v), 1 h, rt (95:5 v/v), 1 h, rt + 200 eq L-Cys·HCl·H2O)

REMOVAL 1 h, rt CONDITIONS

168-171 140, 143, 172 173 REF.

Table 6. Backbone amide protecting groups found in the literature based on p-methoxybenzyl structure.

The same authors, in order to minimize the steric hindrance drawback associated to this group, described in their works the introduction in the sequence of the pre•synthesized Fmoc-AA2-N(Dmob)-AA1-OH building block to allow the synthesis of large peptides.170,171 However, they specified that this dipeptide method is restricted to the AA-Gly dipeptides because when using other amino acids at the N-terminus steric hindrance may preclude their preparation. These dipeptides are commercially available and, as a matter of fact, the Dmb is one of the most used backbone protecting groups, together with pseudoprolines amb Hmb drivatives.

The less studied protecting group with three methoxy units, the 2,4,6-trimethoxybenzyl (Tmb, also named Tmob) (Table 6),172 has also been used as an amide backbone protector for peptides. The incorporation of an extra methoxy group introduces more acid lability to the group compared with its di-substituted analog. Although some comparative studies revealed that Tmob can be removed with only 5% TFA in dichloromethane,143 standard Tmob removal is performed under the same acidic proportion as common cleavages in Fmoc strategies (Table 6). In spite of preference for the di-substitution over the substituted trimethoxy, Tmob resulted in faster acylation than that achieved with Dmb.140,143,172 The facilitated acylation observed for this protecting group is due to a similar hydrogen bonding effect as that described for Hmb (Fig. 8c). The greater the number of methoxy groups in ortho position, the more favored the acylation, thus influencing more the hydrogen bonding than the steric hindrace of the methoxy group. However, bulkiness is the main reason why sometimes neither Dmb nor Tmob are selected as backbone amide protecting groups, although some Dmb- or Tmob-protected AAs are commercially available.

25 General Introduction

A family of photolabile backbone protecting groups, that are also based on para•methoxybenzyl moieties, has also been explored by Kent and co-workers, namely the 2-nitrobenzyl group (2-Nb) and the two methoxy versions (4-methoxy-2-Nb (Table 6) and 4,5-dimethoxy-2-Nb)173 for use in Boc/Bzl SPPS chemistry. They based their studies on the photolytic nitro properties previously reported160,174 and also on the photolytic cleavage analysis.175,176 The introduction of methoxy groups to increase acid lability (prior demonstrated for nitrobenzyl derivative linkers),177 led Kent to confirm that the incorporation of a second methoxy in meta position with respect to the benzyl carbon atom does not favor the photolytic cleavage. It is reasoned because of the extra methoxy position, which represents an electron-withdrawing group with respect to the benzyl carbon atom, and those less electron-rich benzyl carbons lead to decompose less rapid than their more electron-rich analogues. Thereby using the mono-methoxy- substituted nitrobenzyl 4-methoxy-2-Nb analog, they found the most promising backbone amide protecting group. On the other hand, the nitrobenzyl derivatives, besides being cleaved under mild removal conditions, provide the advantage of being acid resistant, allowing to obtain the 4,5-dimethoxy-2-Nb-containing sequences after HF or TFA cleavages. This property enables them to be considered a "safety-catch" protecting group, which has been further studied in the introduction of chapter 2.

2.2.1.1.5. Other structures

Improved alternative protecting groups are the 1-methyl-3-indolylmethyl (MIM)178 or the 3,4-ethylenedioxy-2-thenyl (EDOTn)178 (Table 7), two electron-rich systems, both described by Albericio and collaborators and designed for Fmoc/tBu strategies and maintaining the same range of acid lability as Dmb. The less sterically hindered properties of EDOTn is an advantage, allowing faster acylation than Dmb and thus overcoming the limitations associated with this protector. In 2009, Carpino proposed the dicyclopropylmethyl (Dcpm)179,180 (Table 7) as a backbone protecting group labile to TFA. This group is based on the analog dimethylcyclopropyl (Dmcp)181, previously described by the same author as an amide side-chain protecting group for Asn and Gln, as well as C-terminal amide protector. First, the author described the synthesis of the building block as Fmoc-N(Dcpm)-AA-OH for unhindered AAs and later proposed the pre-synthesized dipeptide building block Fmoc-AA2-N(Dcpm)-AA1-OH to overcome the sterical hindrance,182 a strategy also proposed and mentioned previously for other backbone amide protectors.

26 General Introduction

Two other backbone amide protecting groups not based on benzyl structures are the substituted furfuryl and thienylmethyl,143 both put forward by Quibell in 1999 (Table 7) and designed for Fmoc/tBu SPPS strategy. These two benzyl structures show greater acid lability than Hmb, and only in the case of 5-methoxythienylmethyl derivative the lability is equivalent to the Tmob group. The incorporation of the substituted furfuryl and thienylmethyl moieties is performed by preparing the Fmoc-N(furfuryl derivative)AA-OH or the Fmoc-N(thienyl derivative)-AA-OH. Their coupling onto the peptidyl-resin is performed under common solid-phase conditions, however the most significant limitation of those groups lies in the extremely low yields obtained when the AA is coupled onto the N-(furfuryl/thienyl)peptidyl-resin.

R = H, Me or OMe

derivatives of: MIM EDOTn Dcpm 2-furfuryl 2-thienylmethyl Etom

TFA/H2O/TIS TFA/CH2Cl2/H2O TFA/CH2Cl2/H2O TFA/H2O/TIS (95:5 v/v) TFA/CH2Cl2 or H2O (95:2.5:2.5 v/v), 1 h, rt (95:2.5:2.5 v/v), 1 h, rt (95:3:2 v/v), 1 h, rt R = H (2-3 h), rt (50:50 v/v), 1 h, rt

REMOVAL R = Me or OMe (2 min), rt CONDITIONS

178 178 179, 180, 182 143 183

REF.

Table 7. Backbone amide protecting groups found in the literature that are not based on any common structure.

More recently, the amide backbone protecting group N-alkoxymethyl-based, the ethyloxymethyl (Etom)183 (Table 7) was proposed by Spengler and Albericio to prove its efficiency on enhancing solubility for the Fmoc/tBu SPPS. This group was incorporated into the sequence under standard conditions as dipeptide Alloc-N-(Etom)AA2-N(Etom)-

AA1-OH, previously synthesized. The removal of Etom is carried out under 50% TFA/DCM, however it is also conceivable under mild acidic TFA treatment (5%).

2.2.1.2. Isopeptides

Depsipeptides, also known as O-acyl isopeptides or merely isopeptides, are suitable as solubilizing peptide strategy because the temporary ester bond in the sequence disrupts the hydrogen bond continuity of backbone amides to the same extent as backbone protectors, thus preventing β-sheet interactions and consequently aggregations. This strategy is not mediated by the introduction of an extra protecting group. Indeed, the sequence is synthesized by SPPS, and the ester bond is performed through the hydroxyl group of side-chain of Ser or Thr, instead of performing the

27 General Introduction common amide through the α-amino. The peptide is elongated and then cleaved from the resin, affording the O-acyl isopeptide (Fig. 7).

After the acidic cleavage of the depsipeptide, the unprotected primary amino group of the depsi unit is protonated, thus the intramolecular attack of that amino on the carbonyl of the ester is not produced and it is possible to obtain a stable depsipeptide in solution. In order to achieve the native peptide sequence, once the peptide is released from the resin and unprotected, the last step consists of the O→N-acyl shift, which is mediated by mild basic conditions in aqueous media. Thus, the O→N arrangement occurs through a five-member ring intermediate, reaction controlled by the pH (Fig. 7).

PG2 removal Y = H (Ser) 1) Fmoc removal Y = CH (Thr) 2) Peptide elongation 3

Cleavage pH > 7

O→N-acyl transfer

Figure 7. Isopeptide concept: synthesis by SPPS and the O→N shift in solution to achieve the native sequence. FINAL MÉS GRAN PQ ES MES VERTICAL

The first peptides synthesized by SPPS following the O→N-acyl transfer strategy were published in 1998 by Horikawa and Ohfune184,185, based on earlier studies with peptides synthesized in solution.186–189 However, it was in 2004 when the application of isopeptides as a β-sheet disrupting strategy was exploited, in parallel, by Carpino,190 Kiso,191,192 Mutter193 and Aubagnac194 to synthesize "difficult peptides", where the ester bond was produced through the Ser/Thr-hydroxyl (Fig. 7). Initially, the solid-phase synthesis of these isopeptides was performed in a stepwise manner, including the esterification step; however, some side-reactions were detected after introduction of the depsi unit. These non-desired reactions are associated with racemization during the ester formation or the well-known DKP formation after deprotection of the amino group from the second AA after the depsi unit. Various solutions were proposed in order to minimize or prevent these drawbacks in "difficult peptide" synthesis. Some of them were mainly based on the use of pre-synthesized isodipeptides in solution to prevent racemization;195,196 and others, on the replacement of the Fmoc of the seond AA after the depsi unit by other amino protecting groups labile to milder basic conditions.195,197 The same idea of acyl migration has been reported for O→N- and

28 General Introduction

S→N-acyl isopeptides, depending on the AA involved in the migration step. Therefore, the preparation of new isodipeptides units198,199 has opened up a diversity of building blocks, in the beginning subjected only to Ser/Thr, but now extended to any AA.200 During recent decades, several published syntheses have demonstrated the efficiency of depsipeptide strategies.201,202 In this regard, these strategies favor the cyclization of peptides203 and also peptide elongations by microwave SPPS.204

Although this strategy was merely developed to address the solubility of peptides during the solid-phase elongation, the stability of the isopeptide to cleavage conditions allows to preserve its optimized solubility properties, thus facilitating its manipulation and purification. Other recent variations of the isopeptide method have focused on the O→N-acyl shift step, proposed by Mutter and named "switch•peptides",193 or the "click-peptides"205 proposed by Kiso, where the modulation of the acyl-transfer is mediated by a last selective and controlled reaction, not only by the pH. Thus, in order to favor the stability of isopeptides in solution under neutral conditions, a number of groups have proposed distinct protecting groups for the isopeptide site (PG1 in Fig. 7), assigned as switch elements, that induce the acyl-transfer via a specific trigger. Some examples of proposed switch elements are based on photocleavable protecting groups such as 6-nitroveratryloxycarbonyl (Nvoc), which is removed under photolytic conditions, thus leading to the O→N-acyl transfer.205,206 The azide temporary protecting group, one of the most widely used switch elements, is preserved after cleavage of the peptide, and once the azide is reduced to amino, the O→N arrangement takes place, affording the native sequence.207,208

2.2.2. Solubilization in solution

2.2.2.1. "Pegylation" or glycosylation

The two most studied chemical modifications to increase the aqueous solubility of non-polar peptides are "pegylation" and glycosylation. Both strategies are addressed to enhance solubility, thus facilitating not only the peptide manipulation, but also the peptide reactions in solution. “Pegylation” involves the conjugation of a peptide to a polyethylene glycol (PEG) tag; and glycosylation to a sugar moiety. The "pegylation" strategy was initially introduced in proteins by Davis and collaborators209 in the ’70s and this method was subsequently adopted by other authors for smaller molecules or peptides.210,211 The most significant property associated with the PEG conjugation to a peptide212 is that it provides a substantial increase in solubility of hydrophobic sequences in water. This mechanism relies on the formation of more hydrogen bonds with water through ethylene oxide units213,214 Another advantage, apart from the

29 General Introduction enhancement of solubility, lies on the synthetic facilities to link them to a peptide sequence.215 The most frequent active site in peptides for the attachment of PEG moieties are the amino groups of (α or ε) or the N-terminus of the peptide,216 although new strategies have emerged expanding the versatility of "pegylation" (Fig. 8).217–220 The incorporation of PEG tags into peptide sequences has been extensively studied and some authors have summarized the strategies most used to introduce them in solution and solid-phase peptide synthesis.215,221

PEG O O O O O O O O O O PEG NH N N N N N N N N N PEG

PEG PEG PEG PEG PEG PEG PEG PEG PEG

Figure 8. Peptide positions at which it is possible to introduce a polyethylene glycol chain.

Several examples in the literature have confirmed the efficiency of conjugated PEGs to modify both, the properties of peptides and their architectures.222,223 The PEG moieties most widely used have a molecular weight average of Mw<1000 Da. Moreover, although many kinds of PEG moieties are commercially available, monodisperse PEG units are the most preferred to avoid tough characterizations or even unachievable purifications.216 Various authors have linked the PEG unit through an orthogonal cleavable linker, whereby the PEG moiety temporarily preserves the solubility of certain sequences, thereby facilitating the characterization and purification of the peptide.127 The PEG moiety is then detached from the peptide by a final selective reaction that allows to recover the native sequence. In addition to these appreciated properties, the "pegylation" strategy has additional advantages over other strategies, referred to its lack of toxicity, which has brought about approval by the Food and Drug Administration (FDA). Furthermore, conjugation of drugs to a PEG unit enhances the half-life of these therapeutic agents, thus expanding the number of "pegylated" peptide applications.211,224,225 Recent studies further support the use of "pegylation" strategies as attractive options to modify non-polar peptides in order to increase their 226,227 solubility.

In a parallel manner, glycosylation, in which the peptide sequence is attached to sugar units, has also been developed as a strategy to enhance solubility of peptides and reduce aggregations.228 In contrast to "pegylation", the incorporation of sugar moieties into a sequence is a reaction that occurs in biological systems when glycoproteins are produced by the glycosyltranferase enzymes.229 Sialic acid from carbohydrate moieties

30 General Introduction is responsible for increasing the solubility of the connected hydrophobic molecule, as well as for introducing other therapeutic properties.230

Appropriate orthogonal hydroxyl protecting groups are required for the solution and solid-phase synthesis of glycopeptides. In spite of the difficulties encountered to attach a sugar moiety to a sequence, several authors have further addressed this strategy in recent years (Fig. 9a).231,232 The main linkage of sugar to a peptide sequence is formed through the side-chain of certain AAs, with the modified AA building block generally being previously synthesized and then sequentially introduced into the peptide sequence (Fig. 9b). Depending on the functional group involved in the linkage between the peptide and the saccharide, the sequence afforded is known as N-, O-, or C•glycopeptide. Improved syntheses of glycopeptides both in solution233–235 and on solid-phase86,236–238 have been described even for long peptide sequences. In both strategies, peptides containing conjugated PEG moieties or saccharides require the condensation of large molecules, which may lead to difficult reactions. In this case, the combination of various solubilizing strategies is mandatory.

a)

tBu tBu

N N N O O tBu tBu tBu tBu O

b)

tBu tBu

N H2N

tBu tBu

Figure 9. Two main exemplified strategies to introduce sugar moieties on solid-phase to afford glycopeptides by: (a) stepwise incorporation; and (b) pre-synthesized sugar building block incorporation.

2.2.2.2. Solubilizing short peptide-tags

Given the need to understand the interactions or the behavior of biological systems, recent years have witnessed an increase in the demand for the synthesis of proteins with high purity. The field devoted to the design, development, and optimization of methods to achieve the synthesis of proteins is experiencing constant progress. On the other hand, the use of recombinant methods to achieve proteins has significant bottlenecks with regard to obtaining proteins of considerable purity. In order to overcome purification issues, several authors have supported the strategies based on

31 General Introduction peptide fusion, developed initially in the ’70s and ‘80s. Nowadays, these strategies are probably among those most widely used to purify proteins obtained by recombinant methods. Fusion between a protein and peptide sequence, named peptide tag, is produced by expression systems that introduce these peptides into the N- or C- terminus of target proteins.239 The principal concept underlying such fusion is based on the specific properties conferred by the tag to the large molecule, which allow its purification. After purification of the protein, when necessary removing the previously attached peptide tag the molecules are detached by different mechanisms, for example by the action of specific enzymes.

240,241 The most commonly used tag is the poly-histidine-tag [usually (His)6], amino acid that shows high affinity to metal ions, thus allowing purification by columns composed of a combination of metals that immobilize proteins at a certain pH.242 Other peptide tags bind specifically to certain antibodies, such as FLAG-tag.243 Furthermore, proteins have also been combined with peptides with high affinity to bind streptavidin protein (Strep-tag and SBP-tag),244 where purification is based on affinities to momentarily immobilize the tagged proteins to be isolated from other non-tagged impurities. One of the shortest peptide-tag sequence used for recombinant protein purification is the 245,246 poly-arginine-tag [usually (Arg)5], thus cationic exchange columns are used to isolate proteins carrying this tag.

In parallel, although less studied than peptide tags for recombinant proteins, some short peptide tags have been conjugated to peptide sequences. In this case, the attachment of the tag to a peptide is not addressed to facilitate the peptide separation by column affinity methods, but it is focused on enhancing the solubility of the non•polar peptide targets. Hydrophobic peptide sequences are examples of molecules that would be synthesized following this strategy, especially taking into account the relevance of solubilizing peptides with respect to HPLC characterization or peptide purification. In this group of peptide tags, short sequences are preferred, generally comprising 5-10 residues, rather than small proteins or large peptides, which are extensively selected for protein purification purposes. The short peptide tags used to increase solubility are formed by poly-AAs containing one type of AA (homooligo•peptides), some of these polar and with charged side-chains. Therefore, the basic AAs disposed as poly-arginine and poly-lysine, and the acidic ones sequences poly-glutamic and poly-aspartic are the peptide tags used for this purpose (studied in more detail in the introduction of chapter 3). Synthetic strategies to afford the non•polar peptide connected to the poly-AA tag may be carried out easily on solid•phase because the tag is also formed by AAs. Major examples described in the

32 General Introduction literature are those solubilizing tags introduced on the C-terminus of a target peptide, although some references also report a straightforward synthesis by linking the tag to an AA side-chain.247

The conjugation of a non-polar peptide to a given tag can be achieved in two ways. The two molecules can be linked through a non-hydrolysable bond, such as an amide bond, or through a bifunctional linker that is stable under cleavage conditions but labile to other treatments. Concerning the first method, after the SPPS and the cleavage, the tag persists on the peptide target and it is not possible to detach the two molecules, thus resulting in a permanent conjugation (Fig. 10a). By contrast, in the second approach, the tag is temporarily conjugated to achieve a soluble peptide for further selective release (Fig. 10b). However, after cleavage of the peptide from the resin in both strategies, the peptide and the solubilizing tag remain linked, thus allowing the manipulation of the non-soluble peptide and facilitating the characterization and also the purification. After isolation of the conjugate from any impurities formed during the peptide synthesis, a final step is performed in a temporarily conjugated strategy, whereby the target peptide is released by a specific treatment which removes the linker.

a) b) non-soluble peptide linker solubilizing tail cleavage conditions non-soluble peptide solubilizing tail (linker stable)

cleavage conditions non-soluble peptide linker solubilizing tail

non-soluble peptide solubilizing tail orthogonal removal of solubilizing sequence (linker labile)

non-soluble peptide

Figure 10. Schematic concept of the "solubilizing tail" strategies by conjugation: (a) permanently; and (b) temporarily.

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33 General Introduction

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47

OBJECTIVES

Objectives

The general aim of the present thesis is focused on the development of tools to achieve, by solid-phase, the synthesis of "difficult peptides", as well as the purification of aggregation prone peptide sequences. Evaluation of synthetic strategies and the solubility after the elongation for some selected standard complex sequences will serve of comparison to validate the novel strategies described in this thesis. More specifically, the objectives are the following:

Design and evaluation of several synthetic strategies based on solid-phase to reach one of the well-known "difficult peptides", the RADA-16, as the main aim of chapter 1. Analysis by HPLC of some commercialized samples to define the most significant parameters to be considered when characterizing this kind of peptides. Examination of a post-synthetic RADA-16 procedure to provide a purification tool to achieve aggregating peptides.

Synthesis of a new backbone amide protecting group, the 2-Methoxy-4- methylsulfinylbenzyl (Mmsb), to be later incorporated on solid-phase into "difficult peptide" sequences, as the goal of chapter 2. Elongation of three complex sequences by using the temporary Mmsb group, as a "safety-catch" moiety to improve their syntheses, as well as allowing their purifications.

Enhancing the solubility of non-polar molecules in solution through the conjugation of a short-peptide tag, as the first objective of chapter 3. Evaluation of AA tag type and AA tag spatial disposition influences of linear and branched poly-AA as solubilizing tag sequences. Validation of this strategy by a permanent conjugation of an insoluble drug to a selected solubilizing tag. The second objective relies on studying the efficiency of the Mmsb moiety as a linker to temporarily connect a non-soluble peptide to a poly- AA tag.

New application of Mmsb-OH linker to take part of the orthogonal C-terminal protecting groups. In addition, in chapter 3, evaluation of the proposed protecting group by synthesizing an example of one well-known peptide segment. Stability and lability studies regarding this protecting group to validate its usefulness in peptide fragment condensation in solution.

51

CHAPTER 1. Synthetic Approaches to Reach a "Difficult Peptide"

Introduction

CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

1. The "Difficult Peptide" RADA-16

"Difficult peptides" are characterized by their tendency to form β-sheet inter- or intramolecular interactions during the synthesis, which is considered, at the same time, a challenge. The inaccessibility of functional groups to react affording the amide bond is precluded precisely by those molecular associations. In spite of the synthetic difficulties related to those sequences, their impact in biomedicine has encouraged the researchers to invest great efforts to develop new methodologies to reach them. The appreciated capacity of "difficult peptides" to compose β-sheet secondary structures resides on the sequence transformation from a non-organized (random coil) to a more organized (β-sheet) structure. When this behavior is also produced after the peptide synthesis, thus in solution and under appropriated conditions, these peptides may form aggregates or fibrils, supramolecular stable architectures with interesting properties in the nanomaterials field.

Specifically, those peptide sequences with self-assembling capacity and amphiphilic character are the most valued because the assembly occurs spontaneously in solution and may be modulated by external factors. The facility to manipulate the peptide behavior in amphiphilic peptides has given the possibility to get in aqueous solution initially aggregates, later fibers and subsequently, as a last stage of their organization, hydrogel formations.1 Hydrogels are supramolecular organizations stable enough to be applied in biology, for example as regenerative tissues, drug delivery systems, nanotubular materials, among others.2,3 The wide range of applications in medicinal engineering of amphiphilic peptides have attracted the interest in designing new peptide sequences, not necessarily extracted from natural structures,4 mainly addressed to force its self-assembly.5 Principal designs are focused merely on the synthesis of the subtype of these peptides known as ionic self-assembling peptides, mentioned in the general introduction. In addition to the hydrogen bonds characteristic of the peptide backbone amides, the ionic surfactant-like peptides present the positive-negative ion•ion interactions which are orders of magnitude stronger than the hydrogen bond. Those associations allow the peptides to aggregate in appropriated pH conditions and this is the reason which those ionic sequences form extremely stable hydrogels.

One known example of ionic self-assembling peptide is the called RADA-16, also named RADA-16-I because of belonging to type I (+ − + − + −,...), referring to one•to•one positive-negative alternation within the subtype of ionic self-assembling peptides. The primary structure of RADA-16 is composed by a total of 16 residues with the peculiarity of being composed of repeating the Arg-Ala-Asp-Ala (R-A-D-A)

57 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

sequence four times (Fig. 1a). It belongs to a group of ionic self-assembling structures in which the first described member of those peptides was the EAK-16,6,7 which is a segment of a yeast protein with binding Z-DNA properties. On the contrary, RADA-16 is an artificial de novo designed structure, being all the group of those peptides designed by Zhang and collaborators.8,9 Precisely the alternating and complementary charges that this kind of sequences exhibited under an appropriated pH, is the reason that lead them to adopt a β-sheet conformation in which two faces are defined: the hydrophobic part (Ala residues) and the positive/negative charged core (Arg/Asp residues) (Fig. 1b). Different structural analyses of RADA-16 have been performed to understand the nanofiber formation and its tridimensional organization.10–12 In the most recent published report,13 the RADA-16 has been described as parallel β-sheet strands secondary structure, which are prolonged in the third dimension. These strands are stabilized in one direction by hydrophobic interactions from , and on the other direction, by cation-anion interactions from and aspartates from consecutive peptide chains in parallel position (Fig. 1c).

a)

Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH2

b) c)

Peptide chain Peptide Arg (+) / Asp (-) FIBER GROWING

Figure 1. RADA-16 structure according to Cormier analysis.13 (a) The AA sequence with the corresponding charged side•chains; (b) the tridimensional schematic representation of the disposition of peptide chains when aggregation occurs; and (c) the tridimensional schematic representation of peptide chains orientation when fiber formation occurs. Alanines (in green); arginines (in red); aspartates (in blue); peptide chain direction (in grey); and ionic interactions (in white dots).

In order to obtain the hydrogel material composed by RADA-16 it has been reported that it is required, once the peptide is resuspended in water, the addition of cations or,

58 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

the merely introduction of the peptide into the physiological media, with certain salt concentration to stimulate the peptide to form fibers. Salt addition to induce the self•assembly has been studied and, depending on the sequence, ions from salt could affect the secondary structure or most importantly, favor the peptide-peptide interactions. For example, positively charged face composed of a peptide chain which presents exclusively lysine residues would be filled of the complementary negative ion from the salt composition, being possible the approximation of another positively charged face from other equivalent peptide chains. An analogous effect would occur for negatively charged residues. Thereby, RADA-16, which is formulated in approximately 99.5% of water, has to be administrated in vitro or in vivo by addition of salt solution either some monovalent cations. That peptide was launched in 2002 by the north-American company BD Bioscience as a bioactive hydrogel named PuraMatrix®,14 later commercialized with a different name by other companies and other ranges of purity. In spite of being a peptide not considered a drug because it is not related with a specific interaction in the biological machinery, their intrinsic properties are enough attractive to sell it as a material to be employed in biological systems, even in animals or who knows if will be feasible for humans in a near future. The principal application of RADA-16 is associated to its capacity of formulating this hydrogel to promote the attachment of cells, as it was demonstrated with different mammalian cells. Furthermore, the absence of immune response when RADA-16 is delivered to an animal model, in addition to studies of toxicity and biocompatibility, have opened the application window associated to that peptide to the biological systems. Thus, in recent years, publications related to RADA-16 have appeared for different purposes, specifically focused on the regenerative medicine, which are directly associated with its capacity to induce cell adhesion and migration. It has been proved in vitro, and also in animal models, in systems such as bones, nervous, cardiac or blood (Table 1).8,15–24 In some studies, the RADA-16 hydrogel is applied combined with certain motifs responsible for certain biological activities, thus the self-assembling peptide in that case enhances those bioactivities.25,26 These bioconjugates are specified in table 1 and named as functionalized RADA.

59 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

Related Application Ref. Field

tissue cell adhesion 13, 14 regeneration

brain issue ingeneering tissue 15, 16 (functionalized RADA) regeneration

bone tissue regeneration tissue 17, 18 (functionalized RADA) regeneration

promote angiogenesis tissue 19 (functionalized RADA) regeneration

neurite outgrowth and tissue 20, 21 active synapse formation regeneration

inhibition on growth of human leukemia cells cancer 22 and also in nude mices treatment in vivo

inhibition on growth of breast cancer cells cancer 23 in vitro treatment (functionalized RADA)

Table 1. Some significant published medicinal applications of RADA-16.

The extended list of published reports about RADA-16 applications highlights the importance of obtaining this kind of peptides in the laboratory by optimized protocols. However, from the synthetic point of view, in some cases an ionic self-assembling peptide may, at the same time, belong to the "difficult peptide" group, which is characterized by presenting the aggregation behavior during the synthesis. In particular, RADA-16 is one of those peptides that aggregate in both stages, during and after the synthesis. Therefore, based on Boc/Bzl or Fmoc/tBu SPPS, several approaches have to be considered to reach this kind of sequences. Although the first attempt to synthesize peptides on solid-phase is stepwise, in which AAs are sequentially introduced on the solid support, when the sequences are considered "difficult peptides", standard methodologies are not suitable to reach those peptides with high purities. Other more elaborated strategies have to be addressed to overcome synthetic troubles. When it is not desired to introduce modifications on the sequence or extra protecting groups to overcome the limitations associated with stepwise approaches, an alternative strategy which combines the synthesis on solid-phase and liquid-phase was described some years ago.27 This methodology is known as convergent strategy because the native sequence is rationally and retro-synthetically detached in segments, which in a synthetic flow would be connected in the appropriate manner. Two general and classical convergent methodologies have been described as suitable strategies for synthesis of large sequences or even "difficult peptides": solid-phase fragment

60 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

condensation28,29 and fragment condensation in solution.30 Both of them share the requirement that the condensation of two fragments occurs mandatorily between fully protected sequences. Although several synthetic processes require the evaluation and the synthesis by using both kinds of condensations to get the peptide target,31 herein are detailed the most relevant aspects of the two methodologies which would serve as a guide to an appropriated choice depending on the peptide characteristics.

2. Solid-Phase Fragment Condensation Approach

The convergent strategy based on solid-phase fragment condensation consists on performing the ligation between two peptide segments on the solid support, as its name suggests. In this strategy, the initial rational detachment of the whole peptide in fragments is crucial, thereby SPPS is the tool used to sequentially reach the peptide chains of each fragments, separately. Once a "difficult peptide" or a large sequence is decided to be synthesized by this strategy, firstly it must be divided, considering that the AA that will be in the C-terminal free carboxylic acid of the new fragment would be selected preferably from those not susceptible to racemization, such as Pro or Gly. Racemization is more favored in this strategy as compared to the stepwise approach, because this side-reaction occurs first when the α-hydrogen is abstracted and then the oxazolone intermediate is formed, and is thus reduced or destabilized when Fmoc or Boc are protecting the amino in a single AA sequential coupling. On the contrary, if a peptide fragment is being coupled, the absence of carbamate group close to the activated carboxylic acid and the presence of an acyl group there, promotes the oxazolone formation and subsequently the racemization. Another aspect to have in consideration about the AAs involved in the condensation is related to their hindrance: when no β-branched AAs or when no bulky side-chain protecting groups are selected, the fragment condensation occurs faster and with higher yields, besides side-reactions are precluded (see other requirements in Table 2).

In this strategy, detailed in figure 2, a principal solid support is selected, on which the corresponding C-terminal fragment (whose α-amino is going to react during the condensation) is synthesized stepwise on a solid support which allows to obtain the desired C-terminal functional group. The other peptide segment, the N-terminal fragment (whose carboxylic acid is going to react during the condensation) is synthesized on a certain resin which allows to reach, after the cleavage, a carboxylic acid free in its C-terminus and the functionalized side-chains properly protected. The N-terminal fragment is coupled onto the peptidyl-resin, where the C-terminal fragment had been previously elongated, by fragment condensation under standard conditions

61 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

using the common excess of this fragment and coupling reagents used in stepwise SPPS (1.5-3 eq.). When more fragments have to be coupled onto that peptidyl-resin to complete the target sequence, they are prepared analogously to the first N-terminal fragment, and after removal of the α-amino protecting group from the peptidyl-resin, this new N-terminal fragment is coupled, and so on, and so forth. After all these solid•phase ligations, the last α-amino protector is removed on solid-phase and the final peptide sequence is cleaved from the resin with concomitant removal of the side•chain protecting groups. Only in those cases where only one fragment condensation is required to get the desired target, the last α-amino protector removal could be performed in conjunction with the cleavage step, whenever that group would be labile to cleavage conditions. In contrast, when more fragment condensations are required, the protecting group must be orthogonal, thus being labile to different conditions as compared with the final cleavage.

PG PG PG9 7 5 PG3 PG1 Nt-PG Nt-PG PG 10 PG8 PG6 PG PG4 2 Cleavage under Mild Cond. N-terminal PG Removal

PG PG5 PG9 7 PG3 PG1 OH H2N Nt-PG PG10 O PG PG8 PG6 PG4 2

N-terminal fragment Amide Bond C-terminal fragment Formation

PG PG PG 9 7 5 PG3 PG 1 Nt-PG

PG10 PG PG 8 PG6 PG4 2

Cleavage/PG Removal

Target Peptide

Figure 2. General scheme of the convergent strategy: solid-phase fragment condensation.

The first proposed peptide synthesis by solid-phase fragment condensation was performed in the early '80s and several advantages were attributed to the use of this methodology compared with the classical stepwise strategy.28 In subsequent years this approach was supported by different authors29,32,33 as the most appropriated methodology to synthesize certain peptide sequences by both, the Boc/Bzl or the Fmoc/tBu solid-phase strategies. A reported sequence characteristic where fragment condensation by solid-phase would be profitable, relies on the presence of some repeating AAs fragment along the sequence. This fragment, thereby, would be prepared in large quantity on the same resin and subsequently being introduced on

62 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

the peptidyl-resin, the number of times necessary to reach the final peptide.34 One example of these kind of peptides is the previously mentioned RADA-16. In the literature, several sequences have been synthesized by solid-phase fragment condensation and most importantly, recent published reports35–37 validate this strategy, which remains a reasonable option to be considered when conventional strategies are not able to reach certain peptides.

3. Fragment Condensation in Solution Approach

Parallel to the solid-phase convergent strategy, the fragment condensation in solution was established as an advantageous alternative to build large or "difficult peptide" sequences when limitations associated to firstly described fragment condensation have to be overcome.38,39 These principal obstacles that may be encountered in the solid•phase ligations are related to: (a) equivalent excesses required of the protected N-terminal fragment to carry out the fragment condensation (Fig. 2); and (b) the hindrance of the protected N-terminal fragment (Fig. 2). The first issue involves the necessity to synthesize more quantity of N-terminal fragment, assuming the loss of peptide material. The second limitation becomes severe when fragment condensation is performed on solid-phase instead of in solution phase, thus the solid support nature would not allow the access of bulky molecules. These two aspects are considered reasons that would lead researchers to prefer fragment condensation in solution instead of the solid-phase alternative. However, other essential requirements must be considered when this kind of fragment coupling becomes the strategy of choice (Table 2). Racemization, as well as the hindrance are some of the parameters that must be taken in consideration before deciding the detachment point in the retro-synthetic analysis, in a similar criteria as the solid-phase alternative condensation.40,41

In this strategy, detailed in figure 3, in parallel, two solid supports are properly selected to synthesize independently the N- and C-terminal segments, which subsequently will be coupled. The N-terminal fragment is elongated on a solid support, which, after cleavage, allows to afford a sequence with a carboxylic acid free in its C-terminus, but with a protected N-terminus (semi-permanent protecting group, Nt-PG). The C-terminal fragment is synthesized on a solid support, which, after cleavage, allows to render a sequence with a non-reactive C-terminus (amide or semi-permanent protecting group, Ct-PG), but with an amino free in its N-terminus.

In both peptides, N- and C-terminal fragments, the cleavage conditions to release the sequences before fragment condensation allow all the protecting groups to remain on the side-chains of the AAs, thus only two functional groups (the carboxylic acid and the

63 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

amino), one for each fragment, rest unprotected. Fragment condensation is carried out in a suitable solvent which allow both fragments to be dissolved completely (Table 2). Contrary to solid-phase couplings, a wide range of solvents are available to perform the amide bond formation in solution. Equimolar quantities of the two peptides would be enough to be successful on the ligation step by using standard coupling reagents, or by stronger conditions depending on the particular difficulties encountered.

PG PG5 PG9 7 PG3 PG1 Nt-PG Ct-PG Nt-PG

PG10 PG8 PG6 PG4 PG2 N-terminal PG Removal Cleavage under Mild Cond. Cleavage under Mild Cond.

PG PG5 PG9 7 PG3 PG1 OH H N Ct-PG Nt-PG 2

PG10 O PG PG8 PG6 PG4 2 Amide Bond N-terminal fragment Formation C-terminal fragment

PG9 PG7 PG5 PG3 PG1

Nt-PG Ct-PG

PG10 PG8 PG6 PG PG2 4 Cleavage/PG Removal

Target Peptide

Figure 3. General scheme of the convergent strategy: fragment condensation in solution.

Large peptide sequences, even proteins or "difficult peptides", nowadays are mostly synthesized by following the fragment condensation in solution approach, where the solid-phase syntheses of fragments are separately performed by Boc/Bzl or Fmoc/tBu strategies. Therefore, not only in the past certain complicated sequences were synthesized by this kind of fragment condensation method,42 but also nowadays there are authors which carry on selecting this strategy to reach peptides.43,44 Other peptide fragment condensation strategies in solution have emerged in the recent years,45 such as the well-known native chemical ligation strategy,46 which allows the condensation between unprotected peptide segments enabling the obtaining of complex peptides.47,48

64 CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide" Introduction

Requirements Requirements for Solid-Phase Fragment Condensation for Fragment Condensation in Solution

N-terminal fragment synthesized on resin which N-terminal and C-terminal fragments allows keeping protected side-chains synthesized on resin which allows keeping protected side-chains

low resin loading preferably in C-terminal fragment synthesis high solubility of N-terminal and C-terminal fragments in the same solvent during fragment condensation high solubility in standard SPPS of N-terminal fragment the amino acid on the C-terminus from the N- terminal fragment should not be susceptible to racemization the amino acid on the C-terminus from the N- terminal fragment should not be susceptible to racemization avoid bulky amino acids when choosing the separation point

avoid bulky amino acids when choosing the separation point coupling conditions in fragment condensation should avoid the removal of any PG

coupling conditions in fragment condensation should avoid the removal of any PG

Table 2. Synthetic requirements to perform the fragment condensation strategy on solid-phase31 or in solution.37

Bibliography

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11. Yokoi, H., Kinoshita, T. & Zhang, S. Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc. Natl. Acad. Sci. U. S. A. 12, 8414–8419 (2005). 12. Arosio, P., Owczarz, M., Wu, H., Butté, A. & Morbidelli, M. End-to-end self-assembly of RADA 16-I nanofibrils in aqueous solutions. Biophys. J. 102, 1617–26 (2012). 13. Cormier, A. R., Pang, X., Zimmerman, M. I., Zhou, H.-X. & Paravastu, A. K. Molacular structure of RADA-16-I designer self-assembling peptide nanofibers. ACS Nano 7, 7562–7572 (2014). 14. Zhang, S., Ellis-Behnke, R., Zhao, X. & Spirio, L. Scaffolding in tissue engineering. PuraMatrix: self-assembling peptide nanofiber scaffolds. 217–238 (CRC Press, Boca Raton, FL, 2005). 15. Sieminski, A. L., Semino, C. E., Gong, H. & Kamm, R. D. Primary sequence of ionic self- assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis. J. Biomed. Mater. Res. Part A 87, 494–504 (2008). 16. Zhang, S. et al. Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials 16, 1385–1393 (1995). 17. Cheng, T.-Y., Chen, M.-H., Chang, W.-H., Huang, M.-Y. & Wang, T.-W. Neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering. Biomaterials 34, 2005–2016 (2013). 18. Guo, J. et al. Self-assembling peptide nanofiber scaffold promotes the reconstruction of acutely injured brain. Nanomedicine nanotechnology, Biol. Med. 5, 345–351 (2009). 19. Pan, H. et al. Bone induction by biomimetic PLGA copolymer loaded with a novel synthetic RADA16-P24 peptide in vivo. Mater. Sci. Eng. C 33, 3336–3345 (2013). 20. Semino, C. E. Self-assembling peptides: from bio-inspired materials to bone regeneration. J. Dent. Res. 87, 606–616 (2008). 21. Liu, X. et al. In vivo studies on angiogenic activity of two designer self-assembling peptide scaffold hydrogels in the chicken embryo chorioallantoic membrane. Nanoscale 4, 2720–2727 (2012). 22. Cigognini, D. et al. Evaluation of early and late effects into the acute spinal cord injury of an injectable functionalized self-assembling scaffold. PLoS One 6, 19782–19797 (2011). 23. Tang, C., Shao, X., Sun, B., Huang, W. & Zhao, X. The effect of self-assembling peptide RADA16-I on the growth of human leukemia cells in vitro and in nude mice. Int. J. Mol. Sci. 10, 2136–2145 (2009). 24. Li, J., Zhang, L., Yang, Z. & Zhao, X. Controlled release of paclitaxel from a self- assembling peptide hydrogel formed in situ and antitumor study in vitro. Int. J. Nanomedicine 2143–2153 (2011). 25. Gelain, F., Bottai, D., Vescovi, A. & Zhang, S. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS One 1, 119–130 (2006). 26. Horii, A., Wang, X., Gelain, F. & Zhang, S. Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3- D migration. PLoS One 2, 190–199 (2007). 27. Lee, J. Y. & Bang, D. Challenges in the chemical synthesis of average sized proteins: sequential vs. convergent ligation of multiple peptide fragments. Biopolym. (Peptide Sci.) 94, 441–447 (2010). 28. Pedroso, E. et al. convergent solid phase peptide synthesis. I. Synthesis of protected segments on a hydroxymethylphenyloxymethyl resin using the base labile Fmoc alpha- amine protection. model synthesis of LHRH. Tetrahedron 38, 1183–1192 (1982). 29. Albericio, F., Lloyd-Williams, P. & Giralt, E. Methods in enzymology. Convergent solid- phase peptide synthesis. 289, 313–336 (Academic Press, 1997). 30. Mergler, M. Methods in molecular biology, Vol. 35: peptide synthesis protocols. Synthesis of fully protected peptide fragments. 287–301 (Humana Press Inc., 1994).

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31. Vasileiou, Z., Barlos, K. & Gatos, D. Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain. J. Pept. Sci. United States Am. 15, 824–831 (2009). 32. Lloyd-Williams, P., Albericio, F. & Giralt, E. Convergent solid-phase peptide synthesis. Tetrahedron 49, 11065–11133 (1993). 33. Benz, H. The role of solid-phase fragment condensation (SPFC) in peptide synthesis. Synthesis 4, 337–358 (1994). 34. Dalcol, I., Rabanal, F., Ludevid, M.-D., Albericio, F. & Giralt, E. Convergent solid phase peptide synthesis: an efficient approach to the synthesis od highly repetitive protein domains. J. Org. Chem. 60, 7575–7581 (1995). 35. Johnson, E. C. B., Durek, T. & Kent, S. B. H. Total chemical synthesis, folding, and assay of a small protein on a water-compatible solid support. Angew. Chemie Int. Ed. 45, 3283–3287 (2006). 36. Bayó-Puxan, N., Tulla-Puche, J. & Albericio, F. Oxathiocoraline: lessons to be learned from the synthesis of complex N-methylated depsipeptides. European J. Org. Chem. 2957–2974 (2009). 37. Fagan, V., Toth, I. & Simerska, P. Convergent synthetic methodology for the construction of self-adjuvanting lipopeptide vaccines using a novel carbohydrate scaffold. Beilstein J. Org. Chem. 10, 1741–1748 (2014). 38. Kenner, G. W., Ramage, R. & Sheppard, R. C. Synthesis of a polypeptide chain comprising 129 residues. Strategy and tactincs. Tetrahedron 35, 2767–2769 (1979). 39. Fujii, N. & Yajima, H. Total synthesis of Bovine Pancreatic Ribonuclease A. Part 6. Synthesis of RNase A with full enzymic activity. J. Chem. Soc. Perkin Trans. 1 831–841 (1981). 40. Kimura, T., Takai, M., Masui, Y., Morikawa, T. & Sakakibara, S. Strategy for the synthesis of large peptides: an application to the total synthesis of human [hPTH( 1-84)]. Biopolymers 20, 1823–1832 (1981). 41. Sakakibara, S. Chemical synthesis of proteins in solution. Biopolym. (Peptide Sci.) 51, 279–296 (1999). 42. Athanassopoulos, P., Barlos, K., Gatos, D., Hatzi, O. & Tzavara, C. Application of 2- chlorotrityl chloride in convergent peptide synthesis. Tetrahedron Lett. 36, 5645–5648 (1995). 43. Gracia, C. et al. Convergent approaches for the synthesis of the antitumoral peptide, kahalalide F . Study of orthogonal protecting groups. J. Org. Chem. 71, 7196–7204 (2006). 44. Reddy, P. A., Jones, S. T., Lewin, A. H. & Carroll, F. I. Synthesis of hemopressin peptides by classical solution phase fragment condensation. Int. J. Pept. 1–5 (2012). doi:10.1155/2012/186034 45. Panda, S. S., Hall, C. D., Oliferenko, A. A. & Katritzky, A. R. Traceless chemical ligation from S-, O-, and N-acyl isopeptides. Acc. Chem. Res. 47, 1076–1087 (2014). 46. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. H. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994). 47. Fernández-Tejada, A., Vadola, P. A. & Danishefsky, S. J. Chemical synthesis of the beta- subunit of Human Luteinizing (hLH) and Chorionic Gonadotropin (hCG) glycoprotein hormones. J. Am. Chem. Soc. 136, 8450–8458 (2014). 48. Santhakumar, G. & Payne, R. J. Total synthesis of polydiscamides B, C, and D via a convergent native chemical ligation−oxidation strategy. Org. Lett. 16, 4500–4503 (2014).

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Publication I

______CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I FULL PAPER

DOI: 10.1002/ejoc.201300612

RADA-16: A Tough Peptide – Strategies for Synthesis and Purification

Marta Paradís-Bas,[a,b] Judit Tulla-Puche,[a,b] Aikaterini A. Zompra,*[a,b] and Fernando Albericio*[a,b,c,d]

Keywords: Solid-phase synthesis / Peptides / Self-assembly / Synthetic methods / Nanostructures

The self-assembling capacity of certain molecules can be ex- strategies of synthesis of RADA-16 based on fragment con- ploited for a diverse range of biomedical applications. The densation, and its subsequent purification through optimized ionic complementary peptide RADA-16 is well-known for its methods. Our methodology should prove suitable for the propensity to self-assemble, which derives from its architec- preparation of other peptides prone to self-assembly. tural arrangement. Herein, we describe rational synthetic

Introduction In this study, we chose the well-known self-assembling peptide Ac-(RADA)4-NH2 (RADA-16, Figure 1), a 16 Over the past few years nanobiotechnology and biomateri- amino acid peptide belonging to the family of ionic comple- als based on peptide and protein self-assembly systems have mentary peptides. It comprises repeated segments of hydro- garnered great attention.[1,2] The interest lies chiefly in the phobic (Ala) and hydrophilic (Arg and Asp) groups with possibility of manufacturing new nanostructured scaffolds alternating positive and negative charged amino acid resi- with customizable mechanical properties and biological dues. This structure enables RADA-16 to undergo ordered functions.[3,4] Moreover, peptides and proteins are excellent self-assembly in solution to form nanofibers. model systems for studying biological self-assembly because RADA-16 is highly prone to self-assemble into very they are highly biocompatible and their sequences can be stable and highly organized β-sheet structures that tend to modified to obtain defined molecular properties. Several form hydrogels. It has been extensively used for three-di- types of self-assembling peptides have been systematically mensional cell culture scaffolds as well as for drug delivery studied.[5] This class of biological materials has countless and regenerative medicine.[10–12] Recently, several functional biomedical applications, including tissue regeneration,[6] motifs have been used to generate RADA-16 analogues that drug delivery,[7] protein crystallization,[8] and cellular inter- promote cell adhesion, migration, neurite outgrowth, and nalization.[9] cell differentiation.[13]

Figure 1. Structure of RADA-16.

[a] Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain E-mail: [email protected] The self-assembling capacity of RADA-16, which results [email protected] in a huge range of nanobiotechnology and biomedical engi- Homepage: http://www.irbbarcelona.org neering applications,[14–17] is not only its beauty, but also [b] CIBER-BBN, Networking Center on Bioengineering, Biomaterials and Nanomedicine, the cause of difficulty in both its synthesis in decent yield Barcelona Science Park, 08028 Barcelona, Spain and its purification. Although RADA-16 is commercially [c] Department of Organic Chemistry, University of Barcelona, Marti i Franquès 11, 08028 Barcelona, Spain available and has been synthesized by research groups, the [d] School of Chemistry and Physics, University of KwaZulu- absence of a detailed full chromatographic characteriza- Natal, tion[18] encouraged us to explore combined optimum syn- 4001 Durban, South Africa Supporting information for this article is available on the thetic and purification strategies as well as a convenient WWW under http://dx.doi.org/10.1002/ejoc.201300612. HPLC method for its characterization.[19]

Eur. J. Org. Chem. 2013, 5871–5878 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 5871 71 ______CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I FULL PAPER M. Paradís-Bas, J. Tulla-Puche, A. A. Zompra, F. Albericio

Prompted by the demand for RADA-16 in sufficiently high purity for clinical use, we sought to explore synthetic and chromatographic (HPLC) strategies to obtain this product in high purity. We rationalized that the peculiarity of repeated segments in the RADA-16 sequence would make this peptide an excellent model for developing dif- ferent peptide synthetic strategies to obtain the desired tar- get with maximum possible purity. In this work, rationalized synthetic strategies to obtain RADA-16 have been proposed and carried out to establish the most suitable approach to the synthesis of this peptide with acceptable levels of purity. Stepwise solid-phase syn- thesis, in manually and automatic modes, were first at- tempted, since these are the most common and straightfor- ward ways to synthesize peptides. Two more sophisticated strategies based on fragment condensation approaches (“so- lid-phase fragment condensation” and “fragment condensa- tion in solution”) were performed to evaluate which was the most suitable strategy to reach the peptide target. Approaches outlined herein should prove valuable for other peptide targets that contain repetitive sequences and that undergo self-assembly into other structures.

Results and Discussion Prior to the development of synthetic strategies to obtain RADA-16, an HPLC study of this peptide [in this case, Ac-

(RADA)4-NH2 provided by different companies] was re- quired to optimize the best analytical procedures with which to characterize the self-assembling peptide. Several Figure 2. Comparison of HPLC profiles [conditions: column I HPLC factors were analyzed and optimized,[19] as an exam- heated at 50 °C, eluent based on TFA system, gradient (%B): 0–30] ple, one of the analytical parameters that seriously affects of Ac-(RADA)4-NH2 material provided by one chemical supplier dissolved with H2O/0.1% TFA and analyzed at different concentra- the chromatographic analysis of RADA-16 was the concen- tions: (i) 0.5, (ii) 1, and (iii) 2 mg/mL. tration of peptide injected onto the HPLC column. This factor, which is not typically significant when other peptides are analyzed, is crucial when RADA-16 or other self-as- crude product. Most importantly, impurities associated sembled peptides are studied. The appreciable differences in with this strategy eluted close to the desired product, thus chromatographic profiles of RADA-16 dissolved at concen- complicating the purification, which was already problem- trations of 0.5, 1 and 2 mg/mL show the importance of this atic due to the poor solubility of the peptide (Figure 3, A). parameter (Figure 2). This study allowed us to conclude In fact, one of the principal impurities was derived from that the optimum concentration of HPLC injection for Ac- deletion of the three amino acids Arg, Ala, and Asp, which

(RADA)4-NH2 was ca. 0.5 mg/mL, which reduces aggrega- are side-products that cannot be easily removed, especially tion during application on the HPLC column while still al- when the stepwise approach is hampered by β-sheet forma- lowing peptide purity evaluation. tion. A similar HPLC profile was observed when automated The initial strategy described herein to obtain RADA-16 synthesis was performed (Figure 3, B). was based on stepwise solid-phase syntheses of RADA-16 Since neither manual nor automated stepwise synthesis in manual and automatic modes, using solid-phase peptide gave sufficiently pure peptide, we investigated the solid- synthesis according to Fmoc/tBu chemistry on Chem- phase fragment condensation strategy, whereby presynthe- Matrix resin.[20–23] This resin, which is considered the best sized, protected fragments were sequentially coupled onto suited for this class of peptides, enabled us to overcome the the resin, enabling the peptide sequence to be completed on challenge associated with strongly hydrophobic synthetic solid phase. By following this strategy, side products formed targets, including problems attributed to peptide aggrega- from incomplete couplings are more easily removed because tion or to poor resin solvation. Although after several re- they differ from the peptide by the length of one fragment, couplings, our manual synthesis ultimately afforded the de- rather than by only one amino acid.[24–26] Taking into ac- sired peptide, however, final characterization by HPLC and count that RADA-16 contains the tetrapeptide sequence MALDI-TOF MS revealed a rather poor final purity of the RADA (Arg-Ala-Asp-Ala) in quadruplicate, we decided to

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Figure 3. HPLC profiles of the crude peptide obtained in three different syntheses of RADA-16: (A) manual stepwise solid-phase, (B) automatic stepwise solid-phase, and (C) fragment condensation in solution.

prepare the Fmoc-protected tetrapeptide Fmoc-Arg(Pbf)- propriate solvent for solid-phase coupling. To incorporate Ala-Asp(tBu)-Ala-OH (Fragment F1) on solid-phase, and the synthesized protected fragment F1 onto the resin, care- then link, consecutively, F1 until the desired sequence of ful choice of resin,[29] peptide loading,[30] coupling rea- RADA-16 was complete (Scheme 1). gents,[31,32] and reaction conditions[33] is essential. Again, the ChemMatrix support was chosen for this strategy. At this point, we first tried to incorporate fragment F1 directly onto the polymer support by using a (4 + 4 + 4 + 4 + resin) strategy. However, this approach was unsuccessful and was abandoned. We then turned to the solid-phase fragment condensation (4+4+4+RADA-resin) strategy (Scheme 1); thus, first four amino acids (RADA) were cou- pled stepwise conferring “peptide resin I”. Subsequently, the first fragment F1 was incorporated onto peptide resin I through a double-coupling protocol to afford the resin- bound octapeptide (peptide resin II). The best way to monitor each coupling of F1 onto the resin was to cleave an aliquot of peptide–resin and analyze it by reverse-phase HPLC, which, although more tedious, was essential because standard tests used in stepwise syn- thesis, such as the Kaiser test and the TNBS-test only give a first indication. After Fmoc-removal, the F1 fragment was incorporated (three times) using relatively harsh coupling reagents to afford the dodecapeptide on resin (peptide resin III). HPLC analysis revealed a mixture of products, al- though the desired peptide was obtained in 46 % purity. Af- Scheme 1. Synthesis of Ac-(RADA)4-NH2 (RADA-16) by solid- ter the last fragment F1 incorporation, the Fmoc group was phase fragment condensation. removed and the resulting deprotected peptide was acety- lated and then cleaved from the resin. The main advantage To synthesize the protected fragment F1, with a C-ter- of this strategy was that it minimized formation of side minal carboxyl moiety, we accomplished peptide elongation products that differ from the desired product by only one on CTC resin.[27] Cleavage of the peptide from the resin or two residues, as indicated in narrower peaks observed by under mild acidic conditions provided F1 in high purity reverse-phase HPLC (Figure 3, C). Although for peptides (99%), thus obviating the need for further purification. Al- that are not prone to self-assembly, purification of the crude though the solubility of the obtained product–arequisite material obtained from the fragment coupling strategy was for successful couplings on resin – was unpredictable,[28] for- more convenient than from crude material obtained from a tunately, the target protected tetrapeptide was completely stepwise strategy, in our case, the poor solubility of the soluble in N,N-dimethylformamide (DMF), which is an ap- crude material precluded that.

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Finally, seeking to establish an optimized strategy for reagents and conditions used, and most importantly (4) the RADA-16 synthesis that would ultimately enable synthesis final isolation of the product with concomitant removal of on a larger scale, we explored fragment condensation in any remaining reagents. This strategy has proved to be the solution.[34] Numerous examples of this type of synthesis, strategy of choice for industrial synthesis of several difficult which combines the respective advantages of solid-phase peptide targets[35,36] because it can produce sufficiently pure and solution phase synthesis, have been reported. We chose final product for clinical use. the 8 + 8 strategy (Scheme 2), which required two protected In this work, we synthesized the two protected fragments fragments: the N-terminal fragment (Ac-Arg(Pbf)-Ala- by solid-phase, manually, on medium scale (0.8 mmol) with Asp(tBu)-Ala-Arg(Pbf)-Ala-Asp(tBu)-Ala-OH; Fragment two different resins. In each case, the peptide was cleaved F2) and the C-terminal fragment (H-Arg(Pbf)-Ala- from the resin without affecting the protecting groups. Be-

Asp(tBu)-Ala-Arg(Pbf)-Ala-Asp(tBu)-Ala-NH2; Fragment cause the two fragments were obtained in high purity, no F3). The success of fragment condensation depends on vari- further purification was required. The key point of this ous factors: (1) the solubility and final purity of the pro- strategy was that both fragments were completely soluble tected fragments, (2) the scale of synthesis, (3) the coupling in DMF, because, as previously mentioned, although the solubility of fragments in DMF was mandatory for cou- pling, the solubility of any designed protected fragment is unpredictable. The choice of coupling reagents PyBOP/ DIEA with HOAt as additive, and the use of equimolar amounts of protected fragments, was found to be suitable for this strategy. Phosphonium salts are better than amin- ium salts as activating agents for reactions in which the car- boxylic component was not in excess (e.g., fragment con- densation or cyclization), because they do not react irre- versibly with the amino function, as aminium salts tend to do.[37,38] A clear advantage of fragment condensation in solution over fragment condensation on solid-phase is the quality of the crude peptide obtained. Chromatographic methods are generally unsuitable for purification of peptides such as RADA-16, which are typi- cally insoluble in standard chromatography solvents. Thus, the relatively pure crude material with impurities that are Scheme 2. Synthesis of Ac-(RADA)4-NH2 (RADA-16) by fragment condensation in solution. chemically differentiated from the expected product ob-

Figure 4. HPLC profiles obtained (A) before, (B) during, and (C) after CHCl3/H2O extractions performed in the synthesis of RADA-16 by fragment condensation in solution.

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tained from fragment condensation in solution should be RADA-16 Syntheses purified by washing and precipitation. Washing with tert- Stepwise strategies: We explored manual and automatic stepwise butyl methyl ether helped to remove HOAt, which very solid-phase syntheses of RADA-16, with the double objective of often remains on the peptide. However, although this step optimizing the preparation and also to obtain sufficient amount of can be very efficient in small-scale syntheses, as was the case peptide for use as a reference material. in our attempts, for a larger scale a deeper work-up was Manual Synthesis: RADA-16 was synthesized manually by solid- needed (Figure 4). During this procedure, several impurities phase peptide synthesis by using 9-fluorenylmethoxycarbonyl were removed (Figure 4, B); one of the principal impurities (Fmoc)/tert-butyl (tBu) chemistry on an aminomethyl-ChemMatrix that entered the organic phase had the UV profile of HOAt resin[39] (Matrix Innovation, scale 0.1 mmol, loading: 0.62 mmol/g, (as indicated by photodiode array detector from HPLC 35–100 mesh). Peptide elongation was performed in polypropylene analysis). The expected product was mainly detected in the syringes fitted with a polyethylene porous disk. Solvents and solu- emulsion phase and was isolated as described in detail in ble reagents were removed by suction. The linker Rink-Amide the experimental part and presented in (Figure 4). This pu- (3 equiv., Iris Biotech) was anchored to the resin with N,NЈ-di- rification procedure gave RADA-16 in higher purity (90%; isopropylcarbodiimide (DIPCDI)/1-hydroxybenzotriazole (HOBt) see Figure 4, C) than did either of the stepwise syntheses or (3 equiv. each) in DMF for 12 h. At this point, a positive ninhydrin the solid-phase fragment condensation synthesis. test indicated free amino groups, so the linker reaction was re- peated, this time for 2 h. The Fmoc groups were removed by treat- ment with piperidine/DMF (1:4) in the presence of 0.1 m HOBt (1ϫ 2 min, 2ϫ10 min), which was used to minimize aspartimide formation.[40] The following Fmoc-l-protected amino acid deriva- Conclusions tives were used: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH and Fmoc- Asp(tBu)-OH. Chain extension was carried out by using 5 equiv. It is clear that Ac-(RADA)4-NH2 is a tough peptide to of the amino acid, at each coupling step, in the presence of 2-(6- synthesize and purify. The strategy outlined herein, which chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexa- combines the advantages of solid-phase methodology and fluorophosphate (HCTU) (5 equiv.) and diisopropylethylamine solution chemistry, has allowed the preparation of the tar- (DIEA) (10 equiv.) for 1 h. In case of a positive ninhydrin test, a get peptide with sufficient purity for biological purposes. recoupling was performed, using a fivefold molar excess of the The strategy described herein is based on the solid-phase Fmoc-amino acid, activated essentially in situ by DIPCDI/7-aza-1- preparation of two protected fragments with sufficient pu- hydroxybenzotriazole (HOAt) in DMF for 3 h. Incorporation of 2 3 rity that they do not require purification. Their coupling in the following residues required multiple recouplings: Ala ,Asp, Ala4,Arg5,Ala6,Arg9,Ala10,Asp11, and Ala12. After Fmoc re- solution gives the target product in a purity that can be moval of the last Arg residue, acetylation was performed by treat- resolved further by standard laboratory operations. In ad- ment with Ac2O (10 equiv.) and DIEA (10 equiv.) for 30 min. All dition, a wide range of chromatographic parameters to es- washings of the peptidyl-resin between Fmoc removal, amino acid tablish the optimum HPLC conditions to characterize the coupling and the final acetylation were done with DMF. Once the RADA-16 were analyzed. fully protected peptide had been synthesized, it was subject to This work has shown that, despite the sophistication of global deprotection (removal of the side-chain protecting groups recently developed synthetic strategies for the preparation and cleavage from the resin) by treatment with a mixture of TFA/ of peptides (solid supports, handles, coupling reagents, triisopropylsilane (TIS)/H2O (95:2.5:2.5) for 4 h. The peptide was etc.), the synthesis of self-assembled peptides remains a isolated by precipitation with cold diethyl ether, centrifuged, dis- major challenge. The methodology developed herein should solved in H2O/MeCN/TFA (7:2.8:0.2, starting with neat TFA), and sequentially lyophilized. It was then characterized by HPLC [Col- be of broad applicability to similar peptide targets. umn I; tR = 7.29 min, 75% purity, gradient (%B): 5–25 in 15 min] and MALDI-TOF MS showed the mass of the desired peptide {m/z

calcd. for Ac-(RADA)4-NH2 (C66H113N29O25) 1711.8; found 1712.8 [M + H]+}. Other impurities were also detected from the Experimental Section HPLC analysis and characterized by MALDI-TOF MS: the peak that eluted at t = 6.88 min (peak area: 6.84% purity) corre- General: Analytical HPLC was carried out with an instrument R sponded to a byproduct that lacks the tripeptide RDA {m/z calcd. comprising two solvent delivery pumps, an automatic injector, and for Ac-(RADA)4-NH2 – RAD (C53H91N23O20) 1369.6; found a variable wavelength detector (photodiode array). UV detection 1370.7 [M + H]+}. was performed at 215 and 220 nm. Two HPLC columns were used: column I [C18 column (XTerra, Waters: 4.6ϫ150 mm, 5 μm)] or Automated Synthesis: The automated synthesis was conducted on column II (SunFire, Waters: C18, 3.5 μm 4.6ϫ100 mm). All pep- the same resin used for the manual synthesis (aminomethyl-Chem- tide fragments analyzed from different synthetic strategies of Matrix), with an ABI 433A synthesizer (Applied Biosystems, Fos- RADA-16 were run using linear gradients of two eluents at room ter City), on 0.1 mmol (peptide) scale, following standard Fmoc l temperature (25 °C) and a flow rate of 1.0 mL/min: eluent A (H2O chemistry and using a 10-fold excess of Fmoc-protected -amino + 0.045% TFA) and eluent B (MeCN + 0.036% TFA). HPLC/ES- acids and N-[(1H-benzotriazol-1-yl)-dimethylamino-methylene]-N- MS was performed on a reverse-phase C18 column (3.9ϫ150 mm, methylmethanaminium tetrafluoro-borate N-oxide (TBTU, 0.45 m) 5 μm) using aqueous (+ 0.1% formic acid) and MeCN (+ 0.07% in the presence of HOBt as coupling reagents in DMF. Each depro- formic acid) as eluents. MALDI-TOF MS was carried out with tection step was carried out in 15 min and each coupling step in a Voyager-DETMRP Biosystem, using α-cyano-4-hydroxycinnamic 35 min. Acetylation, cleavage and isolation of the completed pep- acid (ACH) as matrix. tide were done as described for the manual synthesis. The product

Eur. J. Org. Chem. 2013, 5871–5878 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org 5875 75 ______CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I FULL PAPER M. Paradís-Bas, J. Tulla-Puche, A. A. Zompra, F. Albericio was characterized by HPLC [Column I, tR = 7.06 min, 60% purity, Fmoc-(RADA)3-NH2 (C63H94N22O20) 1478.7; found 1479.8 [M + gradient (%B): 5–25 in 15 min] and by MALDI-TOF MS, which H]+}. showed the mass of the desired peptide {m/z calcd. for Ac- Ac-(RADA) -NH : Fragment F1 (1.5 equiv.) was coupled onto (RADA) -NH (C H N O ) 1711.8; found 1712.6 [M + H]+}. 4 2 4 2 66 113 29 25 peptide resin III in two steps: first, with HCTU (1.5 equiv.)/DIEA Solid-Phase Fragment Condensation Strategy (4 + 4 + 4 + RADA- (3 equiv.) for 1 h, which gave a positive ninhydrin test; and then, resin) using DIPCDI/HOAt (1.5 equiv. of each) for 3 h. After Fmoc group removal, the acetylation step was performed by using Ac2O Fragment F1: Fmoc-Arg(Pbf)-Ala-Asp(tBu)-Ala-OH was synthe- (10 equiv.)/DIEA (10 equiv.) for 30 min. An aliquot of peptide resin sized manually by using standard Fmoc protocols on 2-chlorotrityl was taken and then treated with TFA/TIS/H2O (95:2.5:2.5) for 4 h. chloride (CTC) resin (1.55 mmol/g, 10 g). The first Fmoc-Ala-OH The HPLC revealed a mixture of products [Column II; main peak (1.00 mmol/g resin, scale of synthesis 10 mmol) was incorporated tR = 4.24 min, 41% purity, gradient (%B): 5–25 in 8 min], including in the presence of DIEA (10 equiv.), which was added in two por- the desired peptide, RADA-16. MALDI-TOF MS showed the de- tions: first, 1/3 of the volume and, after 10 min, the remaining 2/3. sired product [m/z calcd. for Ac-(RADA)4-NH2 (C66H113N29O25) The mixture was allowed to react for 1 h. Next, a capping step with 1711.8; found 1712.6 (M + H)+], as well as two incorrect mass MeOH (0.4 mL/g) was done. In each deprotection step, removal of signals: one corresponding to Ac-(RADA)3-NH2 (m/z calcd. for the Fmoc group was performed with piperidine/DMF (1:4) C H N O 1298.6; found 1299.8 [M + H]+) and another impu- ϫ ϫ 50 86 22 19 (2 2min,2 10 min). The remaining couplings were done using rity (m/z found 1671.9 [M + H]+) that corresponds to the nonacety- threefold molar excess of the corresponding Fmoc-amino acid acti- lated peptide [H-(RADA)4-NH2 (m/z calcd. 1669.8)]. Based on vated essentially in situ by DIPCDI/HOBt in DMF for 3 h. Be- these results, the acetylation treatment was repeated. The peptide- tween coupling and subsequent deprotection steps, the resin was resin was cleaved using TFA/TIS/H O (95:2.5:2.5) for 4 h. HPLC ϫ ϫ 2 washed with DMF (3 3 min), CH2Cl2 (1 5 min), DMF results still revealed a mixture of products, but the peak at t = ϫ ϫ R (1 3 min), and CH2Cl2 (3 3 min), using 10 mL of solvent/g of 4.94 min had disappeared and the purity of the desired peptide was resin per wash. The complete protected peptide was cleaved from superior to that previously seen in the aliquot [Column II; main ϫ the resin by using TFA/CH2Cl2 (1:99) (6 3 min), and then col- peak tR = 4.26 min, 46% purity, gradient (%B): 5–25 in 8 min]. lected in H2O to avoid side-chain deprotection. After evaporation Fragment Condensation in Solution (8 + 8): Two protected octapep- of CH2Cl2 and lyophilization, the peptide was characterized by tides Ac-Arg(Pbf)-Ala-Asp(tBu)-Ala-Arg(Pbf)-Ala-Asp(tBu)-Ala- HPLC [Column I, tR = 9.14 min, 99% purity, gradient (%B): 40– 100 in 15 min] and by HPLC/ES-MS, which confirmed the desired OH (Fragment F2) and H-Arg(Pbf)-Ala-Asp(tBu)-Ala-Arg(Pbf)- product Fragment F1 {m/z calcd. for Fmoc-Arg(Pbf)-Ala- Ala-Asp(tBu)-Ala-NH2 (Fragment F3) were synthesized separately + on different resins, and then cleaved from the resin with full preser- Asp(tBu)-Ala-OH (C48H63N7O12S) 961.4; found 962.6 [M + H] }. This product was sufficiently pure that it did not require any fur- vation of protecting groups. ther purification. Fragment F2 [Ac-Arg(Pbf)-Ala-Asp(tBu)-Ala-Arg(Pbf)-Ala- Peptide Resin I (RADA-CM Resin): Synthesized on aminomethyl- Asp(tBu)-Ala-OH]: Synthesized manually by using standard Fmoc ChemMatrix resin (0.062 mmol scale) following the same pro- protocols on CTC resin (1.55 mmol/g, 10 g). The first Fmoc-Ala- cedure used for the manual synthesis of RADA-16. At the end, an OH (0.8 mmol/g resin, scale of synthesis 8 mmol) was incorporated using DIEA (10 equiv.) added in two portions: first, 1/3 of the vol- aliquot was taken and treated with TFA/TIS/H2O (95:2.5:2.5) for 2 h. HPLC/ES-MS confirmed the desired (H-Arg-Ala-Asp-Ala- ume and, after 10 min, the remaining 2/3. The mixture was allowed to react for a total of 1 h. Next, a capping step with MeOH NH2) product (m/z calcd. for C16H30N8O6 430.2; found 431 [M + H]+). (0.4 mL/g) was performed. In all deprotection steps, removal of the Fmoc group was carried out with piperidine/DMF (1:4) in the pres- Peptide Resin II ([RADA]2-CM resin): Fragment F1 (3 equiv.), ence of HOBt 0.1 m (1 ϫ2 min, 2ϫ10 min). The remaining cou- which was completely soluble in DMF, was coupled onto peptide plings were accomplished by using threefold molar excess of each resin I in the presence of 1-[bis(dimethylamino)methylene]-1H- Fmoc-amino acid activated in situ by DIPCDI/HOBt in DMF for 1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide 2 h. Washings between coupling and subsequent deprotection steps ϫ ϫ (HATU) (3 equiv.) and DIEA (6 equiv.) for 1 h. Due to a positive were performed with DMF (3 3 min), CH2Cl2 (1 3 min), DMF ϫ ϫ ninhydrin test, a recoupling was performed using fragment F1 (1 3 min) and CH2Cl2 (3 3 min), using 10 mL of solvent/g of (3 equiv.) and equimolar amounts of DIPCDI/HOAt for 3 h. Com- resin per wash. No positive ninhydrin tests were observed at any plete coupling was confirmed by a negative ninhydrin test. An ali- point during the synthesis; therefore, no recouplings were per- quot was taken and treated with TFA/TIS/H2O (95:2.5:2.5) for 2 h. formed. Acetylation was accomplished with Ac2O (10 equiv.)/ HPLC [Column I; tR = 5.88 min, 89% purity, gradient (%B): 20– DIEA (10 equiv.) for 30 min. The protected peptide was cleaved ϫ 80 in 15 min] and HPLC/ES-MS confirmed the desired product from the resin by using TFA/CH2Cl2 (1:99) (8 3 min), and then + (m/z calcd. for C47H67N15O4 1065.5; found 1066.1 [M + H] ). collected in H2O to avoid side-chain deprotection. After evapora- tion of CH Cl , MeCN was added to increase solubility. The prod- Peptide Resin III ([RADA] -CM resin): The Fmoc group of peptide 2 2 3 uct was then lyophilized and characterized by HPLC [Column II; resin II was removed by treatment with piperidine/DMF (1:4) in t = 5.50 min, 98% purity, gradient (%B): 40–100 in 8 min] and by the presence of 0.1 m HOBt. Three couplings were required until a R HPLC/ES-MS, which confirmed the desired product, fragment F2 negative ninhydrin test was obtained. Fragment F1 (1.5 equiv.) was (m/z calcd. for C H N O S 1502.7; found 1504.0 [M + H]+). incorporated onto peptide resin II in three steps: first, using HCTU 68 106 14 20 2 Owing to the high purity (98%) of the final product, no further (1.5 equiv.)/DIEA (3 equiv.) for 1 h; second, with DIPCDI purification was performed. (1.5 equiv.)/HOAt (1.5 equiv.) for 3 h; and finally, with HCTU (1.5 equiv.)/DIEA (3 equiv.) for 1 h. An aliquot was taken and Fragment F3 [H-Arg(Pbf)-Ala-Asp(tBu)-Ala-Arg(Pbf)-Ala- [41] treated with TFA/TIS/H2O (95:2.5:2.5) for 3 h. HPLC [Column II; Asp(tBu)-Ala-NH2]: Synthesized on Sieber amide resin (loading: tR = 3.89 min, 98% purity, gradient (%B): 5–100 in 8 min] and 0.53 mmol/g, 15 g), which allows cleavage of protected peptides MALDI-TOF MS indicated the desired peptide {m/z calcd. for from the resin using 3% TFA. In each deprotection step, removal

5876 www.eurjoc.org © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2013, 5871–5878 76 ______CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I Synthesis and Purification of RADA-16

of the Fmoc group was accomplished with piperidine/DMF (1:4) in the presence of HOBt 0.1 m (1ϫ2min,2ϫ10 min). The cou- [1] S. Zhang, D. M. Marini, W. Hwang, S. Santoso, Curr. Opin. plings were done using a threefold molar excess of each Fmoc- Chem. Biol. 2002, 6, 865–871. amino acid activated by DIPCDI/HOBt (3 equiv. each) in DMF [2] C. J. Bowerman, B. L. Nilsson, Biopolymers (Pept Sci) 2012, for 2 h. Only one recoupling was required: Arg1 (recoupled using 98, 169–184. the same coupling reagents in half-molar quantity for 2 h). The [3] X. Zhao, S. Zhang, Trends Biotechnol. 2004, 22, 470–476. protected peptide was cleaved from the resin by using TFA/CH Cl [4] F. Gelain, A. Horii, S. Zhang, Macromol. Biosci. 2007, 7, 544– 2 2 551. (3:97) (3ϫ3 min), and then H O was added. After evaporation of 2 [5] S. Zhang, in: Encyclopedia of Materials: Science & Technology CH Cl , MeCN was added to increase the solubility of the pro- 2 2 (Eds.: K. H. J. Buschow, R. W. Cahn, M. C. Flemings, B. tected peptide. The product was lyophilized and then characterized Ilschner, E. J. Kramer, S. Mahajan, P. Veyssière), Elsevier, Ox- by HPLC [Column II; tR = 3.21 min, 97% purity, gradient (%B): ford, UK, 2001, 5822–5829. 40–100 in 8 min] and by HPLC/ES-MS, which confirmed the de- [6] R. Fairman, K. Askerfeldt, Curr. Opin. Struct. Biol. 2005, 15, sired structure fragment F3 (m/z calcd. for C66H105N15O18S2 453–463. 1459.7; found 1462 [M + H]+, 731 [M + 2H]+/2). Due to the excel- [7] B. Law, R. Weisslender, C. H. Tung, Biomacromolecules 2006, lent purity of the product, no further purification was required. 7, 1261–1265. [8] J. D. Hartgerink, E. Beniash, S. I. Stupp, Science 2001, 294, Fragment Condensation in Solution: The coupling was carried out 1684–1688. on 0.2 mmol scale. Thus, Fragment F2 (0.2 mmol) was dissolved in [9] S. Pujals, J. Fernandez-Carneado, M. D. Ludevid, E. Giralt, DMF (30 mL). To the solution were added DIEA (2 mmol), then, ChemMedChem 2008, 3, 296–301. added dropwise over 30 min, a solution of 1-benzotriazole-1-yloxy- [10] X. Zhao, S. Zhang, Chem. Soc. Rev. 2006, 35, 1105–1110. tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP)/ [11] K. Hamada, M. Hirose, T. Yamashita, H. Ohgushi, J. Biomed. HOAt (0.2 mmol each) in DMF (43 mL). Fragment F3 (0.2 mmol) Mater. Res. Part A 2008, 84, 128–136. was dissolved in DMF (30 mL) and added slowly to the solution. [12] A. L. Sieminski, C. E. Semino, H. Gong, R. D. Kamm, J. Bi- The reaction mixture was left at room temperature for 16 h and omed. Mater. Res. Part A 2008, 87, 494–504. monitored by HPLC. The DMF was then evaporated in the pres- [13] L. Zhongli, S. Zhang, Chem. Soc. Rev. 2012, 41, 4736–4754. ence of toluene and the product was precipitated by adding cold [14] J. Kisiday, M. Jin, B. Kurz, H. Hung, C. Semino, S. Zhang, A. Grodzinsky, Proc. Natl. Acad. Sci. USA 2002, 99, 9996–10001. tert-butyl methyl ether (3 times). The precipitate was dissolved in [15] C. K. Baig, J. Duhamel, S. Y. Fung, J. Bezaire, P. Chen, J. Am. MeCN/H O (1:1), with sonication (3 min), and then lyophilized. 2 Chem. Soc. 2004, 126, 7522–7532. This process (from the addition of cold tert-butyl methyl ether up [16] T. Scheibel, R. Parthasarathy, G. Sawick, X. M. Lin, H. Jaeger, to the lyophilization) was then repeated twice. The side-chain pro- S. Linquist, Proc. Natl. Acad. Sci. USA 2003, 100, 4527–4532. tecting groups were removed by treatment with TFA/TIS/H2O [17] T. C. Holmes, S. D. Lacalle, X. Su, G. Liu, A. Rich, S. Zhang, (95:2.5:2.5) for 4 h. The peptide was precipitated by adding cold Proc. Natl. Acad. Sci. USA 2000, 97, 6728–6733. diethyl ether, centrifuged, washed with diethyl ether, dissolved in [18] P. K. Ajikumar, R. Lakshminarayanan, B. T. Ong, S. Valiya-

MeCN/H2O (1:1), and then lyophilized. The product was charac- veettil, R. M. Kini, Biomacromolecules 2003, 4, 1321–1326. terized by HPLC, which exhibited one main peak (Column II; tR [19] See the Supporting Information (an accurate HPLC analysis = 4.56 min, 58% purity, gradient (%B): 5–25 in 8 min), and by of six RADA-16 materials provided by different chemical sup- MALDI-TOF MS, which confirmed the desired product, RADA- pliers has been performed). 16. [20] F. García-Martín, M. Quintanar-Audelo, Y. García-Ramos, L. J. Cruz, C. Gravel, R. Furic, S. Côté, J. Tulla-Puche, F. Al- To isolate the desired product and remove any remaining coupling bericio, J. Comb. Chem. 2006, 8, 213–220. reagents or by-products, the following work-up was performed. [21] B. G. De la Torre, A. Jakab, D. Andreu, Int. J. Pept. Res. Ther. Water (0.75 mL) was added to a part of the reaction mixture 2007, 13, 265–270. (200 mg) in DMF (1.5 mL), and a precipitate appeared. The upper [22] T. Kassem, D. Sabatino, X. Jia, X. X. Zhu, W. D. Lubell, Int. J. Pept. Res. Ther. 2009, 15, 211–218. phase was extracted three times with CHCl3, and the combined upper phases together with the pellet/emulsion were centrifuged [23] S. Frutos, J. Tulla-Puche, F. Albericio, E. Giralt, Int. J. Pept. (4000 rpm for 8 min at 4 °C) to isolate the solid and to remove the Res. Ther. 2007, 13, 221–227. [24] Synthesis of cardiodilatin related upper phase. The solid material was washed with cold diethyl ether, K. Nokihara, H. Hellstern, in: peptides by fragment assembly on a polymer support,in:Peptide and then centrifuged twice. The precipitate was then dissolved in Chemistry (Ed.: N. Yanaihara), Protein Research Foundation, MeCN/H2O (1:1) and lyophilized. It was then characterized by Osaka, Japan, 1990, p. 315–320. HPLC, which exhibited a main peak (Column II; tR = 4.41 min, [25] F. Albericio, P. Lloyd-Williams, M. Gairi, G. Jou, C. Celma, 90 % purity, gradient (%B): 5–25 in 8 min). Aliquots from different N. Kneib-Cordonnier, A. Grandas, R. Eritja, E. Pedroso, J. phases during the work-up were taken (CHCl3 phase, emulsion, Van Rietschoten, G. Barany, E. Giralt, in: Innovation and Per- upper phase), and then characterized by HPLC. spectives in Solid Phase Synthesis (Ed.: R. Epton), Andover, UK, 1991, p. 39–47. Supporting Information (see footnote on the first page of this arti- [26] P. Lloyd-Williams, F. Albericio, E. Giralt, Tetrahedron 1993, cle): An accurate HPLC analysis of six RADA-16 materials pro- 49, 11065–11133. vided by different chemical suppliers has been performed. [27] K. Barlos, O. Chatzi, D. Gatos, G. Stavropoulos, Int. J. Pept. Protein Res. 199l, 37, 513–520. [28] R. Nyfeler, in: Methods, in: Molecular Biology (Eds.: M. W. Acknowledgments Pennington, B. M. Dunn), Humana Press, New Jersey, 1994, vol. 35, p. 303–316. This study was partially funded by the Centro de Investigación [29] F. Albericio, M. Pons, E. Pedroso, E. Giralt, J. Org. Chem. Científica y Tecnológica (CICYT) (grant number CTQ2012- 1989, 54, 360–366. 30930), the Generalitat de Catalunya (grant number [30] K. Barlos, D. Gatos, W. Schaefer, Angew. Chem. 1991, 103, 2009SGR1024), and the Institute for Research in Biomedicine (IRB 572–575. Barcelona). [31] D. Hudson, J. Org. Chem. 1988, 53, 617–624.

Eur. J. Org. Chem. 2013, 5871–5878 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org 5877 77 ______CHAPTER 1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I FULL PAPER M. Paradís-Bas, J. Tulla-Puche, A. A. Zompra, F. Albericio [32] Y. Nishiyama, S. Ishizuka, K. Kurita, Tetrahedron Lett. 2001, [37] F. Albericio, M. Cases, J. Alsina, S. A. Triolo, L. Carpino, S. 42, 8789–8791. Kates, Tetrahedron Lett. 1997, 38, 4853–4856. [33] M. Rinnovfi, M. Lebl, M. Soucek, Lett. Pept. Scie. 1999, 6, [38] F. Albericio, J. M. Bofill, A. El-Faham, S. Kates, J. Org. Chem. 15–22. 1998, 63, 9678–9683. [39] S. Côté, WO 2005/012277, 2005. [34] M. Mergler, Methods Mol. Biol. 1994, 35, 287–301. [40] M. Quibell, D. Owen, L. C. Packman, T. Johnson, J. Chem. [35] A. A. Zompra, A. S. Galanis, O. Werbitzky, F. Albericio, Fu- Soc., Chem. Commun. 1994, 2343–2344. ture Med. Chem. 2009, 1, 361–377. [41] P. Sieber, Tetrahedron Lett. 1987, 28, 2107–2110. [36] L. Andersson, L. Blomberg, M. Flegel, L. Lepsa, B. Nilsson, Received: April 17, 2013 M. Verlander, Biopolymers 2000, 55, 227–250. Published Online: August 2, 2013

5878 www.eurjoc.org © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2013, 5871–5878 78 CHAPTER______1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I

Eur. J. Org. Chem. 2013 · © WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2013 · ISSN 1099–0690

SUPPORTING INFORMATION

DOI: 10.1002/ejoc.201300612 Title: RADA-16: A Tough Peptide – Strategies for Synthesis and Purification Author(s): Marta Paradís-Bas, Judit Tulla-Puche, Aikaterini A. Zompra,* Fernando Albericio*

79 CHAPTER______1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I

HPLC ANALYSIS OPTIMIZATION ...... S2 Materials and Methods ...... S2 Evaluation of different HPLC conditions to characterize RADA-16 ...... S2 Results-Discussion ...... S3 HPLC AND ES-MS ANALYSIS OF THE SYNTHESIZED RADA-16 ...... S4

S1 80 CHAPTER______1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I

HPLC ANALYSIS OPTIMIZATION

An accurate HPLC analysis of six RADA-16 materials provided by different chemical suppliers has been performed:

Materials and Methods

Analytical HPLC was carried out on an instrument comprising two solvent delivery pumps, automatic injector and a variable wavelength detector (photodiode array). UV detection was at 215 nm and 220 nm. HPLC columns: Column I (C18 column (XTerra, Waters: 4.6 x 150 mm, 5 µm) or Column II (SunFire, Waters: C18, 3.5μm 4.6 x 100mm) were used at a flow rate of 1.0 mL/min. Liquid chromatography studies of RADA-16 provided by different chemical suppliers were run at linear gradients in three different pairs of eluents A and B (specified its content in every case) at flow rate of 1.0 mL/min: a) TFA based system: eluent A (H2O-0.45% TFA) and eluent B (MeCN-0.036% TFA); b) OSA based system: eluent A [H2O-0.1% 1- Octane Sulfonic Acid Sodium Salt (OSA)] and eluent B (MeCN); c) Perchlorate based system: eluent A (H2O-0.1% (85% H3PO4 aquose)-0.13% sodium perchlorate) and eluent B (MeCN-40% H2O-0.1% (85% H3PO4 aquose)-0.13% sodium perchlorate).

Evaluation of different HPLC conditions to characterize RADA-16

Six different RADA-16 peptides provided by different chemical suppliers were used to optimize the appropriated HPLC conditions to evaluate the quality of this self-assembling peptide. Several chromatographic parameters were tested, and detailed below, in order to provide a wide range of liquid chromatography conditions.

Peptide sample preparation. A RADA-16 sequence (H-(RADA)4-OH) at 1 mg/mL concentration was dissolved in two different conditions [H2O-MeCN (1:1) and H2O-0.1% TFA]. The same linear gradient in column I heated at 50-60ºC was used to evaluate the proper solvent for peptide RADA-16.

Effect of sample concentration. In a 50-60ºC heated column I at the linear gradient and eluent system (1-octane sulfonic acid salt based system), the Ac-(RADA)4-NH2 material was analyzed at different concentrations. Peptide samples at 0.5 mg/mL, 1 mg/mL and 2 mg/mL were injected separately in HPLC with the other chromatographic parameters fixed.

Linear gradient optimization. The RADA-16 sequence H-(RADA)4-OH, was chosen to evaluate one of the most important HPLC factors, the mobile phase gradient. When the purity of a crude peptide is reported, a previous accurate study of optimum gradient is forced to be done. An eluent system based on TFA at different polar linear gradients run at 30 min, was proposed and tested using column I.

Effect of temperature in the HPLC column. When a sample tends to self-assemble or forms aggregations then one of the appropriate approaches to analyze it by HPLC can be to apply temperature to the column (column I). Chromatographic analysis of the same peptide material at 25ºC and in a hot column conditions (50-60ºC) were performed to compare results.

Mobile phase (eluents) selection. Three different eluent systems are proposed in this work to analyze some RADA-16 sequences. The first method is based on TFA in aqueous and MeCN media (system a, detailed above), the second one based on 1-octane sulfonic acid sodium salt in aqueous and MeCN media (system b) and the third one based in sodium perchlorate in aqueous and MeCN media.

S2 81 CHAPTER______1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I

Results-Discussion

A wide range of HPLC conditions have been studied in this work, evaluating the quality of synthetic RADA-16, taking into account the eluent system chosen and the linear gradient as well. Although there are many potential buffers for chromatography, three of them based on TFA, OSA and perchlorate systems were selected in this research. These three buffers differ significantly in ionic strength content and this affects especially the interaction of the peptide with the HPLC column and can explain the differences between profiles. The most appropriate for this self-assembling peptide were those eluents which contain some TFA in H2O and MeCN media [chromatographic profiles show clearly the differences when the eluent parameter is changed (Figure S1).

i) ii) iii)

Figure S1. Evaluation of eluent effect by HPLC analysis of Ac-(RADA)4-NH2 material provided by one chemical supplier dissolved with H2O-0.1% TFA and analyzed at the same HPLC conditions (column I heated at 60ºC, gradient (%B):5-35) but in 3 different eluent systems: H2O and MeCN with TFA (i); H2O and MeCN with perchlorate sodium salt (ii); H2O and MeCN with 1-octane sulfonic acid sodium salt (iii).

Besides, TFA (1%) helps the RADA-16 sample to be dissolved in H2O, thus in this work, that preparation sample protocol was applied to analyze all peptide fragments. HPLC analysis of the same peptide sample when different solvent to solubilize it were tested, reveal that this factor is very important (Figure S2).

i) ii)

Figure S2. Evaluation of RADA-16 peptide sample preparation: HPLC chromatograms (conditions: Column I heated at 50ºC, eluents based on TFA system, gradient (%B): 0-30) of H-(RADA)4-OH material provided by one chemical supplier, dissolved in two different conditions: H2O-MeCN (1:1) (i) and H2O-0.1% TFA (ii).

Characterization by liquid chromatography requires an optimization of the linear gradient in order to distinguish the impurities from the expected material. In this work, a wide range of linear gradient combinations based on TFA eluent system

S3 82 CHAPTER______1: Synthetic Approaches to Reach a "Difficult Peptide"______Publication I allow us to find the appropriated one to analyze RADA-16, which consist on gradient (%B): 5-10 in 30 min. The heating of column at 50-60ºC was another factor should be taken into account, since the higher temperature achieved, less aggregating components of RADA-16 are formed during the analysis, facilitating the identification of some impurities present in the solid material.

HPLC AND ES-MS ANALYSIS OF SYNTHESIZED RADA-16

The RADA-16 synthesized by fragment condensation in solution and obtained after work-up was characterized by HPLC and HPLC-ES-MS:

(M+3)/3

(M+2)/2

Figure S3. HPLC profile and ES-Mass Spectra of RADA-16 synthesized by fragment condensation in solution by HPLC (on the left) and HPLC-ES-MS (on the right). The M corresponds to Ac-(RADA)4-NH2 expected mass.

S4 83

CHAPTER 2. New Backbone Amide Protecting Group

Introduction

CHAPTER 2: A New Backbone Amide Protecting Group Introduction

1. Safety-Catch Structure-Based

1.1. Safety-Catch Concept

In 1971 professor Kenner and collaborators were the first who defined the safety-catch term, as a principle applicable to the synthesis of peptides on solid-phase.1 The same concept was also mentioned by Merrifield and Barany in a book section.2 Specifically, the safety-catch idea described by Kenner establishes that it may be assigned to those molecules that meet the situation where a "...stable bond is eventually labilised at the appropriate moment by a specific chemical modification". Therefore, various kind of structures may be suitable for this description, being the unique requirement that the removal of this linked molecule occurs after a chemoselective transformation.

1.2. Safety-Catch Linkers

The first safety-catch structures for SPPS were proposed as linkers already anchored on the resin, whose advantage lies in the fact that they afford peptide sequences fully protected (the N-, C-terminus and side-chains). These linkers are introduced by reacting the polystyrene resin in order to functionalize it as sulfonamides (Fig. 1a), and are also known, related to its inventor, as Kenner's safety-catch linkers. The peculiarity of the acylsulfonamide peptidyl-resin lies in its stability to nucleophiles, acids or bases. Thus, under these conditions, the linker does not allow the release of the peptide. Particularly, in alkaline media, and due to the pka of the NH group (around 2.5), the deprotonation of the group is more favored than the desired nucleophilic attack. On the contrary, after a chemoselective transformation, such as the alkylation of this NH group, the acylsulfonamide results activated towards nucleophiles because of the enhanced electrophilicity introduced to the carbonyl (Fig. 1a). Specifically, Kenner proposed, for the cleavage, some nucleophiles, such as alkaline hydroxides, the ammonia or the hydrazine to render, respectively, the acid, the amide or the hydrazide C-terminal sequences (Fig. 1a). Although this author developed this novel protocol to render protected peptides containing different C•terminal modifications, one limitation in the Kenner's original idea resides in the last reaction, when poor nucleophiles are not able to cleave the peptide; or when insufficient resin loadings are obtained that favor side-reactions such as isomerization in aspartic or glutamic AAs.

Some years later, Ellman3 and Kiessling,4 independently, proposed improvements based on increasing the electrophilicity of the amide carbonyl towards nucleophiles, by introducing an electron-withdrawing group instead of the methyl moiety (Fig. 1b). In 1996, Ellman and co-workers described an optimized incorporation of the first

89 CHAPTER 2: A New Backbone Amide Protecting Group Introduction

carboxylic group onto the linker to reach higher resin loadings, and also a new alkylating agent which produced cyanomethylation. Both modifications provided significant progress when several substituted amines were used as nucleophiles, even the anilines. In the same study, a new safety•catch functionalized resin (the aliphatic sulfonamide, Fig. 2)5 was presented to facilitate the incorporation of the first carboxylic group onto the resin for those molecules which contained some α-electronegative substituents (such as the N-Fmoc, -Boc AAs), thus situations where the original Kenner's linker rendered poor resin loadings.

a) Labile towards nucleophiles Stable towards nucleophiles Kenner’s safety-catch linker

chemical modification SPPS PG CH2N2 PG N-alkylation

- OH NH3 NH2-NH2 b) ICH2CN

i-Pr2EtN cat. Pd(0)

PG PG PG PG PG

Figure 1. SPPS onto safety-catch sulfonamide linker. (a) Kenner's description which afforded C-terminal peptide derivatives depending on the nucleophile; and (b) two described alternatives to the N-alkylation.

On the other hand, in 2006, Kiessling focused his research on overcoming the troubles of the cyanomethylation reaction, regarding the low yields obtained in the N-alkylation step, as well as the side-reactions associated to the basic conditions required for this reaction. He proposed the N-allylation reaction (instead of N-methylation), thus the allyl substitution may be incorporated by neutral palladium-catalyzed conditions. These mild conditions precludes the non-desired side-products; and again, the intermediate formed is more activated than in the case of the Kenner's linker. Regarding the Ellman and Kiessling optimized acylsulfonamides, several works, most of them published recently, have addressed the efforts to facilitate various difficult, but demanding reactions, mainly peptide head-to-tail cyclizations,6–9 but also to produce C-terminal thioesters,10,11 or even to reach glycopeptides.12

Other examples of activating agents were also developed to improve the yields of the nucleophilic attack and the activation of the carbonyl.13 These mentioned contributions using sulfonamides as safety-catch linkers, together with other kinds of sulfonamide variations, were summarized by Link and Heidler,14 and their versatility has been confirmed for a wide range of organic molecules synthesized,14–17 apart from peptides.

90 CHAPTER 2: A New Backbone Amide Protecting Group Introduction

The stability of those molecules attached to sulfonamide linkers towards the specific alkylation, and also to the cleavage conditions, are two requirements for the use of Kenner-type linkers. In addition to the opportunity to obtain fully protected peptides, these linkers also contribute to expand the number of those already existing handles for Boc/Bzl strategy, which in the beginning were necessarily subjected to arduously HF cleavages. More importantly, the orthogonality of these linkers offer an evident advantage that lies in the wide range of basic, acidic or nucleophilic conditions that may be applied to those molecules connected to these linkers, without causing cleavage.

Apart from the Kenner-type linkers, in 199818 first, and later in 2009,19 Pátek and Lebl summed up the broad diversity of safety-catch linkers described up to date. We have addressed our attention on highlighting those non-based Kenner's linkers which bear the sulfide function, due to the contribution to this field presented in this chapter. Therefore, two types of sulfoxide-based safety-catch linkers may be distinguished: (i) linkers based on the sulfide derivatives studied by Marshall20 (p-mercaptophenol, Fig. 2) and (ii) linkers based on the sulfoxide derivatives proposed by Lebl and co•workers21,22 (the safety-catch amide linker, SCAL, Fig. 2), also studied by Kiso23 (the 4-(2,5-dimethyl-4-methylsulfinylphenyl)-4-hydroxybutanoic acid, DSB, Fig. 2) and, as well as by Undén24 (the 3-(4-Hydroxymethylphenylsulfanyl)propanoic acid, HMPPA, Fig. 2).

Aliphatic sulfonamide p-mercaptophenol HMPPA(O) (Undén) (Ellman) (Marshall)

DSB-resin (Kiso) Aryl sulfonamide (Ellman) Sulfonamide (Kenner) Benzhydryl-based SCAL (Pátek and Lebl)

Figure 2. Some sulfonamide and sulfoxide-based safety-catch linkers described in the literature.

The first type of sulfide-containing linkers (for example, the p-mercaptophenol, Fig. 2) were aimed to reach the synthesis of fully protected peptide sequences. The procedure consists of three steps: the sequence elongation onto the linker, a chemical modification through the oxidation of the sulfide to sulfone, and the cleavage of the peptide by nucleophiles. Thereby, the sulfide function, which preserves the nucleophilic

91 CHAPTER 2: A New Backbone Amide Protecting Group Introduction

attack on the ester moiety, becomes activated towards nucleophiles after being oxidized to the sulfone function, allowing the hydrolysis of the ester. Specifically, Marshall used the glycine sodium salt as nucleophile to obtain a Gly in the C-terminus. The limitation encountered for these linkers resides on the oxidative step, which was not recommended when the sequence contains AAs susceptible to be oxidized, such as Met, Trp, Cys or its cystine derivative. In spite of this drawback, some syntheses of non- peptide-like molecules have been proposed by using the Marshall type linker.25

The second kind of sulfide-containing linkers (for example, the SCAL, the DSB, and the HMPPA(O), Fig. 2) introduces an opposite effect, as they contain the sulfide oxidized to the sulfoxide form. It is worth highlighting that although the HMPPA is introduced onto the resin with the sulfide non-oxidized, this linker is immediately oxidized to sulfoxide after its incorporation on the resin, and before the peptide elongation, being named HMPPA(O) (Fig. 2). The procedure consists of three steps: the sequence elongation onto the linker, a chemical modification through the reduction of the sulfoxide to sulfide, and the cleavage of the peptide by acidolysis. Herein, the linker provides the singularity that the electron-withdrawing effect of the sulfoxide does not allow the peptide being cleaved under acidic conditions. Thereby, this type of safety-catch linkers are stable to hydrolysis under: strong acids, such as the TFA used in Boc/Bzl chemistry, they are also resistant to nucleophiles, or even to those conditions used to remove the N-terminal protector in solid-phase chemical ligations.26 This behavior was not only advantageous for peptides, but also profitable for the obtaining of a large diversity of synthetic organic molecules.27,28 The reduction of the sulfoxide imparts an electron-donating effect that allow the subsequent release of the sequence in acidic media. The peptide cleavage conditions reported by Pátek or Kiso consist of SiCl4/anisole/TFA or (CH3)3SiBr (TMSBr)/thioanisole/TFA cocktails, being the reducing agent and the acid in the same mixture, thus the reduction and the cleavage occur concomitantly (known as reductive acidolysis). On the other hand, some studies, inspired by the previously published 29 30 reduction of by NH4I or by TMSBr, have reported successful results on elongations of peptides which contain disulfide bridges that have been synthesized 28,31 onto SCAL and that have been cleaved by the NH4I/(CH3)2S/TFA cocktail.

1.3. Safety-Catch C-Terminal and Amino Acid Side-Chain Protecting Groups

Although the main research developed on the safety-catch idea was focused on linkers, by exploiting its singular advantage of the stability/lability, several authors have applied this concept also to protecting groups for amino acid side-chains. In this section, only those based on the sulfoxide function have been highlighted. Parallel to

92 CHAPTER 2: A New Backbone Amide Protecting Group Introduction

those explained in the 1.2. section, the removal of the sulfoxide derivative protecting groups contained on the AA side-chain is performed by a reductive acidolysis mediated by the sulfoxide→sulfide chemical modification. One of the most simple protecting group which is suitable for these criteria is the p-(Methylsulfinyl)benzyl (Msib, also named Msob) (Fig. 3b), described in 1988 by Samanen and co-workers32 and based on previous studies based on another application.33 Firstly, Samanen proposed the Msib to protect carboxylic acids, specifically for the C-terminus, and also envisioning its use for Asp and Glu side-chains (Fig. 3a); this last AA protector (OMsob) was proved later by Kiso.34

Professor Kiso also decided to open the applicability of the Msib moiety by incorporating it as an hydroxyl protecting group for Thr or Ser.35 The same author developed a similar protector, modifying it as a carbamate group, the 4-methylsulphinylbenzyloxycarbonyl (Msz) (Fig. 3a). It was conceived for amino groups, not only as N-terminal protector, but also for the ε-amino from Lys side-chain;34 as well as for the protection of the phenol function for Tyr.36 Removal of the Msz, either positioned in the amino or the hydroxyl group, is carried out under the same aforementioned reductive acidolytic conditions, but also under basic treatments (1 M NaOH in MeOH), thus fully orthogonal to Msob removal. More recently, Alewood and co-workers37 have developed the 4,4-dimethylsulfinylbenzhydryl (Msbh) (Fig. 3a) protecting group for Cys protection to form disulfide bonds by a selective protocol, since it is compatible with other cysteine protecting groups.

AA side-chain C-terminal

a) Tyr or Lys Ser or Thr b) Glu or Asp Cys Msib or Msob (Samanen)

Msbh Msz OMsob (Samanen/Kiso) (Alewood) (Kiso) Benzhydryl-based (Pátek and Lebl)

Figure 3. Some sulfoxide-based safety-catch protectors for: (a) the AA side-chains; and (b) the C-terminus.

In the '90s, Professor Lebl and collaborators, followed the same research line as Samanen and designed another sulfoxide-based protector for carboxylic acids (para- substituted benzhydryl-based, Fig. 3b) at the C-terminal position.21 This protecting group was defined as a fruitful method to perform peptide fragment condensation in solution, because the removal of the C-terminal protecting group is carried out after

93 CHAPTER 2: A New Backbone Amide Protecting Group Introduction

the condensation, and those conditions do not affect the peptide integrity, thus being removed concomitantly with other side-chain protectors.

1.4. Safety-Catch Backbone Amide Protecting Groups

The last significant protecting groups based on the safety-catch concept were designed specifically for the backbone amide group in peptides. As it was aforementioned in the general introduction section, the incorporation of these protecting groups provide the advantage of disrupting the β-sheet interactions by enhancing the solubility of peptides during solid-phase, thus allowing the synthesis of "difficult peptides". When the backbone amide protecting group belongs to those defined as safety-catch protectors, another benefit is afforded, which lies in the expanded possibility to treat the sequence under a wide range of conditions, as occur for linkers or AA protectors. The safety-catch backbone amide protecting groups described in the literature up to date, have been mentioned in the general introduction section, and herein are collected in figure 4.

5% N2H4 TFA + acetyl removal Hmb removal

AcHmb

TFA Pd, PhSiH3 + alloc removal Hmb removal

AllocHmb

NH4I/(CH3)2S/TFA + benzoxathiole removal Benzoxathiole derivative

NH4I/(CH3)2S/TFA + Hmsb removal

Hmsb

Figure 4. Safety-catch backbone amide protecting groups and their removal conditions.

94 CHAPTER 2: A New Backbone Amide Protecting Group Introduction

The two first Hmb derivatives (Fig. 4), although they have not been described specifically as safety-catch protectors, they may be included in this concept because the Ac or the Alloc incorporated onto the phenol provides to the Hmb stability under a wide range of conditions during the SPPS. They are suitable for the safety-catch description because only after a certain chemical modification (in these protectors the removal of the Ac or the Alloc group), the Hmb protecting group becomes acid-labile. On the other hand, the last two backbone protectors (Fig. 4) were based on the already mentioned sulfoxide strategy, both groups presents stability in acidic media and when the sulfoxide is reduced to the thioether, subsequently the resulting group becomes cleavable under TFA.

Bibliography

(1) Kenner, G.; McDermott, J.; Sheppard, R. The Safety Catch Principle in Solid Phase Peptide Synthesis. Chem. Commun. 1971, 426, 636–637. (2) Barany, G.; Merrifield, R. B. The Peptides; Gross, E.; Meinhofer, J., Ed.; Vol. 2.; Academic Press: New York, 1979; pp. 1–284. (3) Backes, B.; Virgilio, A.; Ellman, J. Activation Method to Prepare a Highly Reactive Acylsulfonamide “Safety-Catch” Linker for Solid-Phase Synthesis. J. Am. Chem. Soc. 1996, 118, 3055–3056. (4) He, Y.; Wilkins, J. P.; Kiessling, L. L. N-Acylsulfonamide Linker Activation by Pd- Catalyzed Allylation. Org. Lett. 2006, 8, 2483–2485. (5) Backes, B. J.; Ellman, J. a. An Alkanesulfonamide “Safety-Catch” Linker for Solid-Phase Synthesis. J. Org. Chem. 1999, 64, 2322–2330. (6) Yang, L.; Morriello, G. Solid Phase Synthesis of “Head-to-Tail” Cyclic Peptides Using a Sulfonamide “Safety-Catch” Linker: The Cleavage by Cyclization Approach. Tetrahedron Lett. 1999, 40, 8197–8200. (7) Shaheen, F.; Rizvi, T. S.; Musharraf, S. G.; Ganesan, A.; Xiao, K.; Townsend, J. B.; Lam, K. S.; Choudhary, M. I. Solid-Phase Total Synthesis of Cherimolacyclopeptide E and Discovery of More Potent Analogues by Alanine Screening. J. Nat. Prod. 2012, 75, 1882–1887. (8) Lim, H. A.; Tan, L. T.; Chia, C. S. B. Exploring Solution-Phase Cyclization and Sulfamyl Safety-Catch Resin Strategies for the Total Synthesis of the Marine Antimicrobial Cyclic Tetrapeptide cyclo(GSPE). Int. J. Pept. Res. Ther. 2013, 19, 25–31. (9) Kumarn, S.; Chimnoi, N.; Ruchirawat, S. Synthesis of Integerrimide A by an on-Resin Tandem Fmoc-Deprotection-Macrocyclisation Approach. Org. Biomol. Chem. 2013, 11, 7760–7767. (10) Quaderer, R.; Hilvert, D. Improved Synthesis of C-Terminal Peptide Thioesters on “Safety-Catch” Resins Using LiBr/THF. Org. Lett. 2001, 3, 3181–3184. (11) Ollivier, N.; Behr, J.-B.; El-Mahdi, O.; Blanpain, A.; Melnyk, O. Fmoc Solid-Phase Synthesis of Peptide Thioesters Using an Intramolecular N,S-Acyl Shift. Org. Lett. 2005, 7, 2647– 2650. (12) Mezzato, S.; Schaffrath, M.; Unverzagt, C. An Orthogonal Double-Linker Resin Facilitates the Efficient Solid-Phase Synthesis of Complex-Type N-Glycopeptide Thioesters Suitable for Native Chemical Ligation. Angew. Chemie Int. Ed. 2005, 44, 1650–1654. (13) Zohrabi-Kalantari, V.; Heidler, P.; Larsen, T.; Link, A. O,N,N-Trialkylisoureas as Mild Activating Reagents for N-Acylsulfonamide Anchors. Org. Lett. 2005, 7, 5665–5667.

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(14) Heidler, P.; Link, A. N-Acyl-N-Alkyl-Sulfonamide Anchors Derived from Kenner’s Safety- Catch Linker: Powerful Tools in Bioorganic and Medicinal Chemistry. Bioorg. Med. Chem. 2005, 13, 585–599. (15) Maclean, D.; Hale, R.; Chen, M. The Reversed Kenner Linker: a New Safety-Catch Linker for the Preparation of N-Alkyl Sulfonamides. Org. Lett. 2001, 3, 2977–2980. (16) Willoughby, C. A.; Hutchins, S. M.; Rosauer, K. G.; Dhar, M. J.; Chapman, K. T.; Chicchi, G. G.; Sadowski, S.; Weinberg, D. H.; Patel, S.; Malkowitz, L.; Salvo, D.; Pacholok, S. G.; Cheng, K. Combinatorial Synthesis of 3-(amidoalkyl) and 3-(aminoalkyl)-2-Arylindole Derivatives: Discovery of Potent Ligands for a Variety of G-Protein Coupled Receptors. Bioorg. Med. Chem. Lett. 2002, 12, 93–96. (17) Elsawy, M. A.; Martin, L.; Tikhonova, I. G.; Walker, B. Solid Phase Synthesis of Smac/DIABLO-Derived Peptides Using a “Safety-Catch” Resin: Identification of Potent XIAP BIR3 Antagonists. Bioorg. Med. Chem. 2013, 21, 5004–5011. (18) Pátek, M.; Lebl, M. Safety-Catch and Multiply Cleavable Linkers in Solid-Phase Synthesis. Biopolym. (Peptide Sci.) 1998, 47, 353–363. (19) Lebreton, S.; Pátek, M. Safety-Catch Linker Units; Scott, P., Ed.; Wiley.; New York, 2009; pp. 195–220. (20) Marshall, D. L.; Liener, I. E. A Modified Support for the Solid-Phase Peptide Synthesis Which Permits the Synthesis of Protected Peptide Fragments. J. Org. Chem. 1970, 35, 867–868. (21) Pátek, M.; Lebl, M. A Safety-Catch Type of Amide Protecting Group. Tetrahedron Lett. 1990, 31, 5209–5212. (22) Pátek, M.; Lebl, M. Safety-Catch Anchoring Linkage for Synthesis of Peptide Amides by Boc/Fmoc Strategy. Tetrahedron Lett. 1991, 32, 3891–3894. (23) Kiso, Y.; Fukui, T.; Tanaka, S.; Kimura, T.; Akaji, K. A New Reductive Acidolysis Final Deprotection Strategy in Solid Phase Peptide Synthesis. Use of a New Safety-Catch Linker. Tetrahedron Lett. 1994, 35, 3571–3574. (24) Erlandsson, M.; Undén, A. 3-(4-Hydroxymethylphenylsulfanyl)propanoic Acid (HMPPA) as a New Safety Catch Linker in Solid Phase Peptide Synthesis. Tetrahedron Lett. 2006, 47, 5829–5832. (25) Lebreton, S.; Newcombe, N.; Bradley, M. Antibacterial Single-Bead Screening. Tetrahedron 2003, 59, 10213–10222. (26) Brik, A.; Keinan, E.; Dawson, P. E. Protein Synthesis by Solid-Phase Chemical Ligation Using a Safety Catch Linker. J. Org. Chem. 2000, 65, 3829–3835. (27) Tai, C.-H.; Wu, H.-C.; Li, W.-R. Studies on a Novel Safety-Catch Linker Cleaved by Pummerer Rearrangement. Org. Lett. 2004, 6, 2905–2908. (28) Brust, A.; Tickle, A. E. High-Throughput Synthesis of Conopeptides: a Safety-Catch Linker Approach Enabling Disulfide Formation in 96-Well Format. J. Pept. Sci. 2007, 13, 133–141. (29) Nicolás, E.; Vilaseca, M.; Giralt, E. A Study of the Use of NH4I for the Reduction of Methionine Sulfoxide in Peptides Containing Cysteine and Cystine. Tetrahedron 1995, 51, 5701–5710. (30) Beck, W.; Jung, G. Convenient Reduction of S-Oxides in Synthetic Peptides, Lipopeptides and Peptide Libraries. Lett. Pept. Sci. 1994, 1, 31–37. (31) Brust, A.; Wang, C.-I. A.; Daly, N. L.; Kennerly, J.; Sadeghi, M.; Christie, M. J.; Lewis, R. J.; Mobli, M.; Alewood, P. F. Vicinal Disulfide Constrained Cyclic Peptidomimetics: a Turn Mimetic Scaffold Targeting the Transporter. Angew. Chemie 2013, 125, 12242–12245. (32) Samanen, J. M.; Brandeis, E. The p-(methylsulfinyl) Benzyl Group: A Trifluoroacetic Acid (TFA)-Stable Carboxyl-Protecting Group Readily Convertible to a TFA-Labile Group. J. Org. Chem. 1988, 53, 561–569.

96 CHAPTER 2: A New Backbone Amide Protecting Group Introduction

(33) Johnson, B. J.; Jacobs, P. M. A New Carboxy-Protecting Group for Peptide Synthesis and Its Direct Conversion into an Activated Ester Suitable for Peptide Formation: 4- (methylthio)phenyl and 4-(methylsulphonyl)phenyl Esters. Chem. Commun. 1968, 73– 75. (34) Kiso, Y.; Kimura, T.; Yoshida, M. A New Class of Amino Protecting Group Removable by Reductive Acidolysis: The 4-Methylsulphinylbenzyloxycarbonyl (Msz) Group. J. Chem. Soc. Chem. Commun. 1989, 1511–1513. (35) Kimura, T.; Fukui, T.; Tanaka, S.; Akaji, K.; Kiso, Y. A Reductive Acidolysis Final Deprotection Strategy in Solid Phase Peptide Synthesis Based on Safety-Catch Protection. Chem. Pharm. Bull. 1997, 45, 18–26. (36) Kiso, Y.; Tanaka, S.; Kimura, T.; Itoh, H.; Akaji, K. New Hydroxyl Protecting Groups of a Safety-Catch Type Removable by Reductive Acidolysis. Chem. Pharm. Bull. 1991, 39, 3097–3099. (37) Dekan, Z.; Mobli, M.; Pennington, M. W.; Fung, E.; Nemeth, E.; Alewood, P. F. Total Synthesis of Human Hepcidin through Regioselective Disulfide-Bond Formation by Using the Safety-Catch Cysteine Protecting Group 4,4-Dimethylsulfinylbenzhydryl. Angew. Chemie Int. Ed. 2014, 53, 2931–2934.

97

Publication II

CHAPTER______2: A New Backbone Amide Protecting Group ______Publication II

DOI: 10.1002/chem.201403668 Full Paper

& Solid-Phase Synthesis 2-Methoxy-4-methylsulfinylbenzyl: A Backbone Amide Safety- Catch Protecting Group for the Synthesis and Purification of Difficult Peptide Sequences Marta Parads-Bas,[a, b] Judit Tulla-Puche,*[a, b] and Fernando Albericio*[a, b, c, d]

Abstract: The use of 2-methoxy-4-methylsulfinylbenzyl lected as a model to optimize the new protecting group

(Mmsb) as a new backbone amide-protecting group that strategy. Second, the complex, bioactive Ac-(RADA)4-NH2 se- acts as a safety-catch structure is proposed. Mmsb, which is quence was chosen to validate this methodology. The im- stable during the elongation of the sequence and trifluoro- provements in solid-phase peptide synthesis combined with acetic acid-mediated cleavage from the resin, improves the the enhanced solubility of the quasi-unprotected peptides, synthetic process as well as the properties of the quasi-un- as compared with standard sequences, made it possible to

protected peptide. Mmsb offers the possibility of purifying obtain purified Ac-(RADA)4-NH2. To extend the scope of the and characterizing complex peptide sequences, and renders approach, the challenging Ab(1-42) peptide was synthesized

the target peptide after NH4I/TFA treatment and subsequent and purified in a similar manner. The proposed Mmsb ether precipitation to remove the cleaved Mmsb moiety. strategy opens up the possibility of synthesizing other

First, the “difficult peptide” sequence H-(Ala)10-NH2 was se- challenging small proteins.

Introduction These kinds of peptides are characterized by incomplete re- moval of the amino protecting group and poor incorporation Solid-phase peptide synthesis (SPPS) is nowadays the most of the protected amino acids at some stage of the synthesis, common strategy used to synthesize biopolymers in high yield which prevents peptide elongation even after repeating and purity. This approach has been made possible thanks to reaction treatment. It is accepted that these difficulties are due great efforts of the scientific community to develop a wide to the formation of peptide secondary structure, such as range of functionalized resins,[1] coupling reagents,[2–5] and or- b-sheets.[12,13] During the synthesis, these intra- or inter- thogonal protecting groups.[6,7] After extensive fine-tuning molecular interactions, which are usually sequence-dependent, over the last 50 years, one would assume that chemists would spontaneously form aggregations. These assemblies are have all the tools necessary to prepare a short sequence of attributed to the formation of the backbone amide hydrogen any nature. However, this is not the case. The so-called “diffi- bond with carbonyl groups of peptide chains also bound to cult sequences”, which are short sequences that are apparently the resin. unreachable targets,[8–11] continue to pose a challenge. Self-interactions require the presence of the NH of the pep- tide bond, therefore, Pro-containing peptide sequences are rel- atively free of this problem. In this regard, Mutter described a revolutionary concept in protecting group strategy: the [a] M. Parads-Bas, Dr. J. Tulla-Puche, Prof. F. Albericio pseudoproline. This concept converts Ser, Thr, and Cys into Institute for Research in Biomedicine (IRB Barcelona) Baldiri Reixac 10, Barcelona, 08028 (Spain) their five-membered ring dimethyl derivatives, thi/ox- Fax: (+ 34)93-4037126 azolidines, which, during the final global deprotection, render E-mail: [email protected] the native residues.[14,15] Following the same idea of masking [email protected] the presence of the NH moiety, Kiso, Carpino, and Beyermann [b] M. Parads-Bas, Dr. J. Tulla-Puche, Prof. F. Albericio independently proposed the synthesis of Ser and Thr depsi- CIBER-BBN, Networking Centre on Bioengineering Biomaterials and Nanomedicine, Barcelona Science Park peptides, which are more manageable (soluble building Baldiri Reixac 10, Barcelona, 08028 (Spain) blocks) and can be converted into the corresponding peptides [16–19] [c] Prof. F. Albericio in basic medium. Department of Organic Chemistry, University of Barcelona Parallel to the work of Mutter, Sheppard and co-workers put Mart i Franqus 1-11, Barcelona, 08028 (Spain) forward the use of an amide-protecting group, N-(2-hydroxy-4- [d] Prof. F. Albericio methoxybenzyl) (Hmb).[20,21] However, during activation, the School of Chemistry & Physics, University of Kwazulu-Natal a a University Road, Westville, X54001, Durban, 4001 (South Africa) building block N -Fmoc-N -(Hmb)amino acid forms a 4,5-di- Supporting information for this article is available on the WWW under hydro-8-methoxy-1,4-benzoxapin-2(3H)-one, which shows poor http://dx.doi.org/10.1002/chem.201403668. reactivity.[22] Thus, these residues should be incorporated in the

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form of O,N-bis(Fmoc) derivatives. Once these building blocks have been introduced, and after removal of both Fmoc groups, the coupling of the incoming amino acid is facilitated through a seven-membered ring O!N transacylation mecha- nism. In addition, the same authors proposed the use of a so- called “reversible backbone amide” protecting group, N-(3- methylsulfinyl-4-methoxy-6-hydroxybenzyl) (SiMB). In addition Figure 1. The concept of the new backbone amide-protecting group (Mmsb). to the free hydroxyl group, SiMB contains an electron-with- drawing sulfoxide substituent para to the OH, with the unique sophisticated linkers based on the same principle have also purpose of enhancing the acylation step.[23] Global deprotec- been used for SPPS.[36] tion is carried out through a reductive acidolysis to convert Here, we used the Mmsb group to prepare three “difficult the sulfoxide into the thioether and then allow the amide pro- sequences”. After TFA global deprotection, the Mmsb-contain- tecting group to be cleaved by trifluoroacetic acid (TFA). Simi- ing peptides were characterized and purified, when necessary, larly, the so-called “reversible” backbone protecting groups before performing a reductive TFA treatment, which reduces based on the nitro group, which is cleavable under photolysis, the sulfoxide to thioether and concomitantly removes it in were proposed by Kent and co-workers.[24] a one-pot reaction.

During recent years, our group has focused on the synthesis First, the H-(Ala)10-NH2 sequence was selected as a model to of these “difficult sequences”. In this regard, we have used di- study and optimize the new synthetic methodology proposed

methoxybenzyl (Dmb) and developed 1-methyl-3-indolylmeth- herein. Second, two interesting bioactive peptides, Ac-(RADA)4- [25] yl (MIM) and 3,4-ethylenedioxy-2-thenyl (EDOTn) to reduce NH2 (RADA-16) and Ab(1-42), both with a high tendency to intra- and inter-chain interaction, as well as prevent side reac- self-assemble, were synthesized for the validation of the Mmsb tions associated with the presence of the NH group, such as strategy, established in the first model. The first sequence is aspartimides[26–28] and internal diketopiperazine formation.[29,30] a challenging peptide for SPPS because of its hydrophobicity The design of the last two protecting groups mentioned has and its high tendency to form secondary structures that jeop- shown some advantages over benzyl derivatives. However, we ardize the deprotection of the amino function and also acyla- have focused our efforts on a more global solution for the syn- tion.[37] This peptide has been widely used to assay novel syn- thesis and on the characterization and purification of complex thetic approaches.[38, 39] The second peptide, RADA-16, is of in- sequences that is not limited to Ser/Thr/Cys but valid for terest because of its biomedical applications,[40,41] but it is a large number of residues. Here we describe a “safety-catch” again associated with a complex synthesis.[42] In this case, ag- backbone amide-protecting group[31] that is compatible not gregation is related to its high content of alternating positively only with the 9-fluorenylmethoxycarbonyl (Fmoc) strategy, but and negatively charged amino acids. Finally, we addressed the also with TFA treatment, with the latter being used to detach synthesis of the molecule considered to be responsible for the peptide from the resin and remove the classical side-chain amyloid formation in Alzheimer’s disease, the extensively protecting groups. The synthesis of a quasi-protected peptide, studied Ab(1-42).[43,44] The synthesis of this peptide is which still bears the safety-catch protecting group, facilitates challenging[18,45,46] because this molecule shows a strong the characterization and purification of the peptide, and allows tendency to form aggregates, which directly affects the the target free-peptide to be rendered after a final and clean elongation sequence and, at the same time, its solubility, thus chemical treatment. complicating its purification. The Fmoc SPPS strategy is based on the use of a temporary protecting group that is removed by a base (piperidine) and Results and Discussion cleavage upon global deprotection (by TFA), therefore, we chose not introduce any extra chemical reaction. We envisaged To introduce the new Mmsb backbone amide-protecting that the pair alkylsulfoxide/thioether would serve our require- group into the peptide sequence, the corresponding Fmoc de- ments. Thus, the electron-withdrawing sulfoxide group intro- rivative (Fmoc-N(Mmsb)-Ala-OH in our case) was prepared. The duces stability to a benzyl moiety, and it can be easily reduced synthesis of this building block was accomplished in five steps to the corresponding electron-donating thioether, which con- (Scheme 1, see the Supporting Information for details).[47] Com- fers acid lability to the same benzyl group. Specifically, 2-me- mercially available 3-methoxythiophenol (1) was methylated in thoxy-4-methylsulfinylbenzyl (Mmsb), which is stable to TFA, practically quantitative yield with iodomethane (MeI) by careful was reduced to 2-methoxy-4-methylthiobenzyl (Mmtb), which dropwise addition of triethylamine to prevent dialkylation. is labile to TFA, thus perfectly matching our requirements Next, alkylated product 2 was formylated by reaction with (Figure 1). freshly prepared Vilsmeier reagent,[48] which rendered the two The Mmsb moiety has been used for the preparation of the possible isomers. 2-Methoxy-4-methylthiobenzaldehyde (3) corresponding carbamate-based amine protection and inte- was isolated (48% yield) after purification by reverse-phase grated as a linker for the preparation of peptide acids.[32] The chromatography on a C18 column. The reductive amination of demethoxy (p-methylsulfinylbenzyl) version was also used as aldehyde 3 with the amine of the unprotected alanine was [33] [34] a carboxyl protecting group and as a linker, and its reached in a one-step reaction with NaBH3CN in dioxane/H2O corresponding carbamate-based amine protection.[35] More (1:1).[25] N(Mmtb)-Ala-OH (4) was protected with the Fmoc

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Ala-OH was introduced with only 1.5 equiv (AA/coupling re- agent), and the best conditions for the key incorporation of the incoming residue consisted of three couplings with Fmoc- Ala-OH (10 equiv), DIPCDI (10 equiv), and OxymaPure (10 equiv) at 45 8C, with MeCN/N,N-dimethylformamide (DMF) (3:1) as solvent. Once again, we observed that MeCN is a suita- ble solvent for incorporations onto hindered amino acids.[50] Except for the coupling of the incoming Ala on the N-Mmsb residue, no double couplings were performed. However, the three peptide elongations were checked by the Kaiser test[51] after each coupling. The tests revealed that synthesis of the standard peptide took place with incomplete incorporations of Scheme 1. Synthesis of Fmoc-N(Mmsb)-Ala-OH. O/N=overnight. the last four Ala residues. The two modified Mmsb-containing peptides were cleaved

from the resin by using TFA/CH2Cl2 (19:1), rendering both se- group by using a slight excess of Fmoc-OSu in basic media to quences with similar purity (Figures S3 and S4 in the Support- afford Fmoc-N(Mmtb)-Ala-OH (5), which was easily purified by ing Information), and the total stability of the Mmsb group

reverse-phase chromatography on a C18 column to remove was confirmed by MS analysis (m/z calcd for C39H63N11O12S: the excess of unreacted alanine during the reductive amination 909.44; found: 932.60 [M+ Na]+ (8) and 932.45 [M+ Na]+ (9)).

step. Finally, oxidation of 5 with H2O2 rendered Fmoc- Finally, we used MS analysis to study the reductive acidolysis N(Mmsb)-Ala-OH (6) in excellent purity (98.0 %) and without of the Mmsb-containing peptides caused by treatment with

further purification; this building block was used directly for NH4I/TFA (Figure 3 and Figure S12 in the Supporting Informa- SPPS. The overall yield to convert 1 into 6 was 19%. tion). It was found that the Mmsb group was completely re-

First, we tested our synthetic strategy on H-(Ala)10-NH2.As moved after 30 min in the case of 8 and after 2 h in the case a control, the standard peptide was also synthesized with only of 9. This deprotection was accomplished without the need for [23,32] the use of Fmoc-Ala-OH (7; Figure 2). Two modified H-(Ala)10- scavengers (some articles report the use of Me2S) .

NH2 sequences were synthesized with one Fmoc-N(Mmsb)-Ala- One proposed solution to prevent the main impurity detect- OH at positions 6 (8) and 8 (9), respectively. ed in both modified Mmsb-containing peptide syntheses (9% The three peptides were synthesized on Rink amide-poly- in 8 and 6% in 9) associated with the truncated peptide after styrene (PS) as a solid support and N,N’-diisopropylcarbodi- coupling of the N-Mmsb residue involved preparing the dipep- imide (DIPCDI)/ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma- tide (in this case Fmoc-Ala-N(Mmsb)-Ala-OH) in solution (see Pure)[49] as coupling reagent. In this regard, Fmoc-N(Mmsb)- the Supporting Information for details). This strategy was used

Figure 2. Structures of the synthesized peptides.

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Figure 4. MALDI-TOF MS and solubility assays of H-(Ala)10-NH2 crude pep- tides synthesized: a) by using the common Ala, 7, and b) by using the modi- 1 fied Fmoc-N(Mmsb)-Ala-OH, 8. Concentration: 1 mgmL in H2O. Mass im- purities assigned (red label), mass expected peptide (green label).

a b-sheet structure profile (minimum at 215 nm), characteristic of self-assembling sequences, whereas the two modified pep- tides (8 and 9) showed a random coil structure (minimum at 200 nm). These data suggested the involvement of the Mmsb in the secondary structure of this peptide, possibly explaining

the synthetic improvements observed in modified H-(Ala)10-

NH2. Encouraged by the results obtained in this peptide model using the new method, we explored the application of Mmsb Figure 3. Monitoring of the Mmsb group removal by MALDI-TOF MS spectra. to other “difficult peptides”. Starting material (Mmsb-protected H-(Ala) -NH ) 8 is highlighted in blue, 10 2 We recently reported that an acceptable quality of RADA-16 whereas the reaction intermediate (Mmtb-protected H-(Ala)10-NH2) and final

product (unprotected H-(Ala)10-NH2) are shown in orange and green, respec- can be obtained only by coupling two protected octapeptides tively. in solution.[42] In that study, we showed that neither the pro- tected nor unprotected RADA-16 can be purified by chroma-

for the synthesis of modified H-(Ala)10-NH2 8, which showed an tography. We learned that the difficulty arises after the incor- increased purity and the absence of the mentioned impurity poration of 4–5 amino acids. Thus, two syntheses, a standard (Figure S8 in the Supporting Information), thus confirming an approach to overcome the steric hindrance associated with the N(Mmsb) residue. The main conclusion drawn from these analyses is that the influence of the backbone protection has an effect that is apparent even six amino acids further down the backbone. Furthermore, in these two syntheses (8 and 9), no peptides lacking Ala residues were detected (Figure 4b and Figure S4b in the Supporting Information). The standard pep- tide synthesized with common Ala (7) showed four sequences lacking Ala residues, as shown by MS (Figure 4a); therefore, these results are consistent with those obtained from the qual- itative Kaiser test. In addition, the increase in solubility of the

modified sequences in common peptide solvents (H2OorH2O/ MeCN, 1:1) was extraordinary (Figure 4b). In addition to HPLC and MS analysis of standard and modi- fied H-(Ala) -NH , and by means of circular dichroism (CD), we 10 2 m explored the effect on secondary structure when one Mmsb Figure 5. CD spectrum of a 25 m solution of synthesized H-(Ala)10-NH2 in H2O/2,2,2-trifluoroethanol (TFE) (9:1) at pH 5.2 of standard sequence 7 (solid unit was introduced into the peptide sequence (Figure 5). The curve) and modified sequences 8 and 9 (dotted and dashed curves, respec- CD spectrum of the standard H-(Ala)10-NH2 peptide 7 showed tively).

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Figure 6. HPLC/MALDI-TOF MS/solubility assays of crude RADA-16 peptides synthesized: a) by using the common amino acids, and b) by using the modi- 1 fied Fmoc-N(Mmsb)-Ala-OH in position 12. Concentration: 1 mgmL in H2O. Mass impurities assigned (red label); mass expected peptide (green label).

RADA-16 (10) and a modified version (11), were carried out on Rink amide-ChemMatrix resin,[52] by using DIPCDI/OxymaPure as coupling reagent. On the modified peptide, Fmoc-N(Mmsb)- Ala-OH was introduced in position 12 (11). Peptide elongations were again checked qualitatively by the Kaiser test. Although no double couplings were performed, the synthesis of the standard RADA-16 showed, by Kaiser test, incomplete cou- plings in the last four residues. Fmoc-N(Mmsb)-Ala-OH was in-

troduced as in the modified H-(Ala)10-NH2 examples. The cou- pling of the incoming Fmoc-Asp(tBu)-OH was studied (Table 1 in the Supporting Information), concluding that the confluence of a MeCN/DMF (3:1) solvent mixture and three couplings at 458C using DIPCDI and OxymaPure, was once again crucial for satisfactory introduction of the incoming residue. The final global deprotection/cleavage required prolonged acid treatment because of the high content of Pbf side-chain protecting groups of Arg [4 h with TFA/triisopropylsilane (TIS)/

H2O (38:1:1)]. Under these more demanding conditions, the acid-stability of the Mmsb group was confirmed. Although no significant differences between the two RADA-16 peptides were detected by HPLC (Figure 6), the MALDI-TOF MS analysis of these compounds revealed that standard RADA-16 con- tained several deletions (Figure 6a and Figure S2 in the Sup- porint Information; already anticipated by the Kaiser test re- sults during synthesis), whereas the modified RADA-16 had Figure 7. HPLC chromatograms of modified RADA-16 peptide: top) after none. Careful study of the HPLC and MS results concluded that peptide synthesis, middle) after purification, and bottom) final peptide after the modified RADA-16 showed less than 1% of the impurity of removing Mmsb group followed by desalting.

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Figure 8. Analysis and characterization of synthesized modified Ab(1-42): HPLC and MALDI-TOF MS of a) crude Mmsb-protected peptide after synthesis, b) pu- 1 rified Mmsb-protected peptide, c) unprotected peptide after Mmsb removal, and d) solubility of Ab(1-42) at 1 mg mL in H2O of the Mmsb-protected ana- logue (orange label) and final unprotected Ab(1-42) (blue label).

truncated peptide (5-mer, uncompleted incorporation of Asp assembly (12, 21AA), we manually inserted Fmoc-N(Mmsb)-Ala- after coupling of modified N(Mmsb) residue; Figure 6b and OH, followed by the incoming Fmoc-Phe-OH. Both amino acids Figure S5 in the Supporting Information).[53] Most importantly, were coupled by following the same conditions described pre- the solubility of the modified RADA-16 crude peptide in water viously for other modified peptides. Once the elongation of was significantly higher than that of the standard peptide the peptide was complete, the peptidyl resin was treated with

(Figure 6). TFA/TIS/H2O (38:1:1) for 2 h, again confirming the acid-stability The lower solubility of the standard RADA-16, combined of Mmsb. Although the crude peptide obtained showed poor with the poor HPLC resolution of the expected peptide, for purity (35%; Figure 8a and Figures S6 and S7 in the Support- which the impurities eluted close to the desired product, pre- ing Information) by HPLC analysis on a C4 column, our princi- vented its purification. In contrast, the greater solubility of pal aim was to enhance the solubility of Ab(1-42) and facilitate Mmsb-containing RADA-16 allowed its isolation. The final its purification. modified peptide was purified by HPLC analysis on a reverse- The Mmsb-containing Ab(1-42) was isolated by HPLC using phase C18 column, and the Mmsb-protected peptide was iso- the analogous semi-preparative C4 column. The enhanced sol-

lated in excellent purity (99.9 %) (m/z calcd for C75H123N29O27S: ubility of the modified peptide (Figure 8d) allowed its purifica- 1893.89, found: 1894.69 [M+H]+ ; Figure 7 and Figure S9 in tion (Figure 8b, and Figures S10 and S11 in the Supporting In- the Supporting Information). To remove the Mmsb group, the formation). Mmsb removal was completed after 2 h, checked purified peptide was treated with a concentration of by HPLC and MS (MALDI-TOF and HRMS-ESI), with both tech- 1 1mgmL NH4I/TFA and the backbone protecting group was niques confirming the synthesis of the natural Ab(1-42) in 90% completely removed after 45 min, with only a final desalting purity and with the expected mass (HRMS-ESI m/z calcd for + step (see the Supporting Information for details) required to C212H321N55O62S2 : 4693.3098, found: 1129.3273 [M+ 4H] /4, reach the pure target RADA-16 peptide (Figure 7 and Fig- 903.6643 [M+ 5H]+/5 and 753.3872 [M+6H]+ /6; Figure 8c and ure S14 in the Supporting Information). Figures S15 and S16 in the Supporting Information). Inspired by the possibility of purifying RADA-16, we moved onto a second level, extending the application of the Mmsb Conclusion backbone-protecting group to the Ab(1-42) sequence. This amyloidogenic peptide was produced on an aminomethyl Mmsb is a new backbone amide-protecting group that is used resin with a previous incorporation of 3-(4-hydroxymethylphe- in a safety-catch strategy. This new group is stable during noxy)propionic acid as a linker in an automatic synthesizer global deprotection conditions. Mmsb represents a new con- with non-optimized conditions. In the middle of the sequence cept for the synthesis and purification of “difficult peptides”. Its

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presence in a peptide confers several advantages over a stan- chain-protecting groups: TFA/CH2Cl2 (19:1) for 1 h (H-(Ala)10-NH2, 7) dard synthesis. Thus, the elongation of the peptide sequence and TFA/TIS/H2O (38:1:1) for 4 h (RADA-16, 10). The mixture was takes place smoothly and with better yields because of the dis- partially evaporated under reduced pressure and the peptides ruption of the secondary structure. Furthermore, its use could were precipitated with cold diethyl ether. The liquid layer was re- moved by centrifugation and the solid was washed with cold di- prevent side-reactions, such as aspartimide and diketopipera- ethyl ether to give white solids that were dissolved in H2O/MeCN zine formation, while facilitating others, such as cyclization. We (1:1) and lyophilized. The crude peptides were analyzed by HPLC have demonstrated that the presence of only one Mmsb and MALDI-TOF MS (see Figures S1 and S2 in the Supporting molecule favors the incorporation of at least 10 amino acids in Information).

the case of H-(Ala)10-NH2 and RADA-16. In addition, the Mmsb- protected peptides are much more soluble than standard pep- tides, thus allowing better HPLC resolution and, most Synthesis of modified peptide sequences importantly, their purification. The improved solubility could be explained by the absence of secondary structure and also The modified peptide sequences [two analogues modified by the polarity of the sulfoxide. The strategy described here H-(Ala)10-NH2 : one in position 6 (8), the second in position 8 (9); and the modified Ac-(RADA) -NH in position 12 (11)] were synthe- could be applied to any amino acid other than Ala. Preparation 4 2 sized on the same resins described in case of each standard in solution of dipeptides (equivalent to pseudoprolines or dep- peptide. The resin washings, commercial amino acid couplings, sipeptides) is advisable when using Mmsb-hindered amino and Fmoc removal cycles were performed by following the same acids, so as to facilitate incorporation in the peptide sequence. protocol described for the standard sequences synthetic methodol- The removal of Mmsb takes place under conditions that do ogies. The Kaiser test was used to detect which amino acid was not damage the peptide and allows the desired peptide to be not completely coupled (neither in 8, 9, nor 11 were uncompleted obtained after only one filtration. Notably, our results on the couplings detected). No double couplings were performed except synthesis of Ab(1-42), for which only one Mmsb group was in- in the case of the incoming amino acid to H-N(Mmsb)-Ala-OH. The synthesis of modified Ab(1–42) (12) was carried out by an automat- troduced within 42 AA residues, support the proposed synthet- ic microwave peptide synthesizer (Discover, CEM corporation). The ic approach. The results show that this novel synthetic tech- synthesis was performed with an Aminomethyl ChemMatrix resin nique enhances the manipulation of the peptide, especially (160mg, 0.1mmol, 0.62mmolg1) with a previous incorporation of with regard to its solubility and purification. We envisage that 3-(4-hydroxymethylphenoxy)-propionic acid as a linker, coupled this technology will have broad use not only for the synthesis with the same system described in standard sequences. Commer- of “difficult peptides”, but also for the purification of a large cial and common Fmoc-l-AA(PG)-OH amino acids were used. The range of other peptides. coupling system consisted of N-[(1H-benzotriazol-1-yl)-(dimethyla- mino)methylene] N-methylmethanaminium hexafluorophosphate (HBTU) with N,N-diisopropylethylamine (DIEA) and DMF as a solvent Experimental Section and the Fmoc removal was performed with piperidine/DMF (1:4). Synthesis of standard peptide sequences

H-(Ala)10-NH2 (7) and RADA-16 (10) peptides were synthesized on Incorporation of the synthesized building block Fmoc- Rink-Amide AM Polystyrene resin (250 mg, 0.12 mmol, N(Mmsb)-Ala-OH 0.48 mmolg1) and Rink-Amide AM ChemMatrix resin (100 mg, 0.053 mmol, 0.53 mmolg1), respectively. Polystyrene resin was After Fmoc removal of the previous amino acid, the Fmoc- N(Mmsb)-Ala-OH (1.5 equiv) was coupled by using DIPCDI conditioned by washing with DMF (31 min) and CH2Cl2 (3 1 min), and ChemMatrix resin was conditioned by initial washing (1.5 equiv) and OxymaPure (1.5 equiv) in DMF for 1h. Then, wash- with TFA/CH Cl (1:99) (51 min), CH Cl (51 min), DIEA/CH Cl ings with DMF (3 1min) and CH2Cl2 (31min) were performed 2 2 2 2 2 2 and Kaiser test confirmed the complete incorporation of the AA. (1:19) (51 min) and CH2Cl2 (5 1 min). Commercial amino acids were coupled in both synthesis as follow: Fmoc-l-AA(PG)-OH (3 equiv), DIPCDI (3 equiv), and OxymaPure (3 equiv) in DMF, with a 5 min preactivation and with a total coupling time of 1 h. After Incorporation of the incoming Fmoc-AA-OH to H-N(Mmsb)- every coupling, the resin was washed with DMF (31 min) and Ala-OH CH2Cl2 (31 min). Then, a Kaiser test was carried out to check which amino acid was not coupled completely [4 AA: Ala1-Ala4 in Modified RADA-16 (11) was selected to find the best coupling con- 9 6 4 1 the case of H-(Ala)10-NH2 (7); and 4 AA: Ala , Ala , Ala , Arg in the ditions to introduce the incoming amino acid (see Table S1 in the case of RADA-16 (10)]. No double couplings were performed inde- Supporting Information) and the extension of this reaction was pendently of the test results. Next, the Fmoc removal was per- evaluated by HPLC analysis of the crude peptide after an aliquot of formed with piperidine/DMF (1:4) (25 mLg1 resin, 11 min, 2 peptidyl-resin was treated with a cleavage cocktail. The solvent

5 min), followed by resin washings with DMF (31 min), CH2Cl2 was then evaporated and the peptide was precipitated with cold (31 min), and DMF (31 min). The cycle of AA coupling/Fmoc re- diethyl ether. Amino acid coupling consecutive to H-N(Mmsb)-Ala- moval was performed until complete elongation of peptides, in- OH was accomplished by following the same conditions used in all cluding the last Fmoc removal. In the case of RADA-16 (10), a final modified peptide sequences, at 458C for 2h in DMF/MeCN (1:2),

step of acetylation was accomplished by Ac2O (10 equiv) with DIEA with the coupling reagent system DIPCDI (10 equiv) and Oxyma- (10 equiv) in DMF for 30 min. Final resin washings were carried out Pure (10 equiv) for Fmoc-Phe-OH (10 equiv). A total of three

with DMF (3 1 min) and CH2Cl2 (31 min). The peptides were sub- consecutive couplings (using the same coupling conditions) were jected to cleavage treatment depending on the amino acid side performed.

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Cleavage/global deprotection of side-chain protecting [10] R. C. de L. Milton, S. C. F. Milton, P. A. Adams, J. Am. Chem. Soc. 1990, groups of modified peptides 112, 6039 –6046. [11] J. Bedford, C. Hyde, T. Johnson, W. Jun, D. Owen, M. Quibell, R. C. Shep- After all the peptide elongation, cleavage, and post-cleavage treat- pard, Int. J. Pept. Protein Res. 1992, 40, 300 –307. ments were performed by following specified conditions depend- [12] M. Mutter, V. N. R. Pillai, H. Anzinger, E. Bayer, C. Toniolo, in Proc. 16th ing on the amino acid side chain-protecting groups nature, the Eur. Pept. Symp. (Ed.: K. Brunfeldt), Scriptor, Copenhagen, 1981, pp. 660 –665. peptides were subjected to cleavage treatment TFA/CH2Cl2 (19:1) for 1h (8 and 9), TFA/TIS/H O (95:2.5:2.5) for 4h (11) and TFA/TIS/ [13] S. M. Meister, S. B. H. Kent, in Proc. to 8th Am. Pept. Symp. (Eds.: V. J. 2 Hruby, D. H. Rich), Pierce Chemical Company Co., Rockford, 1983, H O (95:2.5:2.5) for 2h (12). The mixture was partially evaporated 2 pp. 103 –106. under reduced pressure and the peptides were precipitated with [14] T. Haack, M. Mutter, Tetrahedron Lett. 1992, 33, 1589 –1592. cold diethyl ether. The liquid layer was removed by centrifugation [15] G.-A. Cremer, H. Tariq, A. F. Delmas, J. Pept. Sci. 2006, 12, 437–442. and the solid was washed with cold diethyl ether to give white [16] Y. Sohma, M. Sasaki, Z. Ziora, N. Takahashi, T. Kimura, Y. Hayashi, Y. Kiso, solids that were dissolved in H2O/MeCN (1:1) and lyophilized to in Proc. to 18th Am. Pept. Symp. (Eds.: M. Chorev, T. K. Sawyer), American afford the modified sequences. The crude peptides were analyzed Peptide Society, Boston, 2003, pp. 67–68. by HPLC and MALDI-TOF MS. Peptides were characterized by HPLC [17] Y. Sohma, M. Sasaki, Y. Hayashi, T. Kimura, Y. Kiso, Chem. Commun. 2004, and MALDI-TOF MS (see Figures S3–S7 in the Supporting Infor- 124– 125. mation). [18] L. A. Carpino, E. Krause, C. D. Sferdean, M. Schmann, H. Fabian, M. Bi- enert, M. Beyermann, Tetrahedron Lett. 2004, 45, 7519 – 7523. [19] I. Coin, P. Schmieder, M. Bienert, M. Beyermann, J. Pept. Sci. 2008, 14, Removal of the Mmsb amide protecting group 299– 306. [20] T. Johnson, M. Quibell, D. Owen, R. C. Sheppard, J. Chem. Soc. Chem. Lyophilized modified crude peptides 8, 9, 11, and 12 (1–5 mg) Commun. 1993, 369 –372. 1 were dissolved in neat TFA (concentration 1 mgmL ) in a round- [21] M. Liley, T. Johnson, Tetrahedron Lett. 2000, 41, 3983 –3985. bottomed flask with constant stirring at RT. Ammonium iodine [22] E. Nicols, M. Pujades, J. Bacardit, E. Giralt, F. Albericio, Tetrahedron Lett. (30 equiv) was then added to the reaction mixture and the reaction 1997, 38, 2317 –2320. was monitored by HPLC (an aliquot was taken from the reaction [23] J. Howe, M. Quibell, T. Johnson, Tetrahedron Lett. 2000, 41, 3997 –4001. [24] E. C. B. Johnson, S. B. H. Kent, Chem. Commun. 2006, 1557 –1559. mixture, the TFA was removed by N2 (g), and the peptide was pre- [25] A. Isidro-Llobet, X. Just-Baringo, M. Alvarez, F. Albericio, Biopolymers cipitated in diethyl ether and redissolved in H2O/MeCN (1:1)). When MALDI-TOF MS analysis revealed the completion of the reac- 2008, 90, 444– 449. tion, removal of the iodine byproduct was carried out by filtration [26] J. P. Tam, M. W. Riemen, R. B. Merrifield, Pept. Res. 1988, 1, 6– 18. [27] E. Nicols, E. Pedroso, E. Giralt, Tetrahedron Lett. 1989, 30, 497– 500. of the reaction mixture. The majority of volatiles were removed [28] Y. Yang, W. V. Sweeney, K. Schneider, S. Thçrnqvist, B. T. Chait, J. P. Tam, under reduced pressure and the precipitation of the peptide was Tetrahedron Lett. 1994, 35, 9689 –9692. performed by transferring the peptide solution dropwise to cold [29] W. Lunkenheimer, H. Zahn, Justus Liebigs Ann. Chem. 1970, 740, 1– 17. diethyl ether. After three times centrifugation and diethyl ether [30] B. F. Gisin, R. B. Merrifield, J. Am. Chem. Soc. 1972, 94, 3102 –3106. washings, the solid was lyophilized and the peptides were ana- [31] Our definition of safety-catch protecting group is based on that used lyzed by MALDI-TOF MS (see Figures S12–16 in the Supporting by Ptek and Lebl for the same type of linkers: “provides high stability Information). during the synthesis, but capable of chemoselective modification and subsequent labilization for cleavage step”.[54] [32] S. Thennarasu, C.-F. Liu, Tetrahedron Lett. 2010, 51, 3218 –3220. Acknowledgements [33] J. M. Samanen, E. Brandeis, J. Org. Chem. 1988, 53, 561– 569. [34] M. Erlandsson, A. Undn, Tetrahedron Lett. 2006, 47, 5829 – 5832. [35] Y. Kiso, T. Kimura, M. Yoshida, J. Chem. Soc. Chem. Commun. 1989, The work was partially supported by CICYT (CTQ2012–30930), 1511–1513. the Generalitat de Catalunya (2014SGR 137), and by the Insti- [36] S. Lebreton, M. Ptek in Linker Strategies in Solid-Phase Organic Synthe- tute for Research in Biomedicine (Spain). We really thank Dr. sis, (Ed.: P. Scott), John Wiley & Sons, New York, 2009, pp. 195-220. Natalia Carulla for her helpful advice regarding the manipula- [37] S. A. Kates, N. A. Sole, M. Beyermann, G. Barany, F. Albericio, Pept. Res. 1996, 9, 106 –113. tion of Ab(1–42). [38] D. Englebretsen, P. Alewood, Tetrahedron Lett. 1996, 37, 8431-8434. [39] S. Abdel Rahman, A. El-Kafrawy, A. Hattaba, M. F. Anwer, Amino Acids 2007, 33, 531– 536. Keywords: peptides · protecting groups · solid-phase [40] K. Hamada, M. Hirose, T. Yamashita, H. Ohgushi, J. Biomed. Mater. Res. synthesis · synthesis design · synthetic methods Part A 2008, 84, 128 –136. [41] Z. Luo, S. Zhang, Chem. Soc. Rev. 2012, 41, 4736 – 4754. [1] M. Gngora-Bentez, J. Tulla-Puche, F. Albericio, ACS Comb. Sci. 2013, 15, [42] M. Parads-Bas, J. Tulla-Puche, A. A. Zompra, F. Albericio, Eur. J. Org. 217– 228. Chem. 2013, 5871 –5878. [2] F. Albericio, L. A. Carpino, Methods Enzymol. 1997, 289, 104 –126. [43] C. Haass, D. J. Selkoe, Nat. Rev. Mol. Cell Biol. 2007, 8, 101– 112. [3] S.-Y. Han, Y.-A. Kim, Tetrahedron 2004, 60, 2447 –2467. [44] S.-H. Yen, W.-K. Liu, F. L. Hall, S.-D. Yan, D. Stern, D. W. Dickson, Neuro- [4] T. I. Al-Warhi, H. M. A. Al-Hazimi, A. El-Faham, J. Saudi Chem. Soc. 2012, biol. Aging 1995, 16, 381–387. 16, 97– 116. [45] A. B. Clippingdale, M. Macris, J. D. Wade, C. J. Barrow, J. Pept. Res. 1999, [5] A. El-Faham, F. Albericio, Chem. Rev. 2011, 111, 6557 – 6602. 53, 665– 672. [6] L. A. Carpino, J. Am. Chem. Soc. 1957, 79, 98– 101. [46] Y. Sohma, M. Sasaki, Y. Hayashi, T. Kimura, Y. Kiso, Tetrahedron Lett. 2004, [7] A. Isidro-Llobet, M. Alvarez, F. Albericio, Chem. Rev. 2009, 109, 2455 – 45, 5965 –5968. 2504. [47] Conditions for the alkylation and formylation steps were slightly adapt- [8] S. B. H. Kent, in Proc. 9th Am. Pept. Symp. (Eds.: K. D. Deber, C. M. Hruby, ed from those described in ref. [32]. V. J. Kopple), Pierce Chemical Company Co., Rockford, 1985, pp. 407 – [48] U. C. Dyer, D. A. Henderson, M. B. Mitchell, P. D. Tiffin, Org. Process Res. 414. Dev. 2002, 6, 311– 316. [9] E. Atherton, R. C. Sheppard, Solid Phase Peptide Synthesis-A Practical Ap- [49] R. Subirs-Funosas, R. Prohens, R. Barbas, A. El-Faham, F. Albericio, proach, Oxford University Press, Oxford, England, 1989. Chemystry - A Eur. J. 2009, 15, 9394 –9403.

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[50] G. A. Acosta, M. del Fresno, M. Paradis-Bas, M. Rigau-DeLlobet, S. Ct, [53] The very low percentage of 5-mer impurity showed quite different M. Royo, F. Albericio, J. Pept. Sci. 2009, 15, 629 –633. chromatographic properties compared with the expected peptide, [51] E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Anal. Biochem. 1970, which facilitates the purification step. 34, 595– 598. [54] M. Ptek, M. Lebl, Biopolymers 1998, 47, 353 –363. [52] S. Ct, New Polyether Based Monomers and Highly Cross-Linked Am- phiphile Resins, 2005, WO 2005/012277 A1. Received: May 23, 2014 Published online on October 3, 2014

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Supporting Information

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2014

Marta Paradís-Bas,[a, b] Judit Tulla-Puche,*[a, b] and Fernando Albericio*[a, b, c, d]

chem_201403668_sm_miscellaneous_information.pdf

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Supporting Information

2-Methoxy-4-methylsulfinylbenzyl, a backbone amide safety-catch protecting group for synthesis and purification of difficult peptide sequences

Marta Paradís-Bas,[a, b] Judit Tulla-Puche*[a, b] and Fernando Albericio*[a, b, c, d]

[a]Institute for Research in Biomedicine (IRB Barcelona) Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain; Fax: (+34)93-4037126; E-mail: [email protected], [email protected]. [b]CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain [c]Department of Organic Chemistry, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain [d]School of Chemistry & Physics, University of Kwazulu-Natal, Durban 4001, South Africa

Materials and General Methods...... 2

Building Block synthesis...... 5

A) Fmoc-N(Mmsb)-Ala-OH...... 5

B) Fmoc-Ala-N(Mmsb)-Ala-OH...... 9

Solid-Phase Peptide Synthesis...... 12

Synthesis of Modified Peptide Sequences...... 12

Analysis of Peptide Crudes...... 14

A) Analysis of Standard Peptide Sequences...... 14

B) Analysis of Modified Peptide Sequences...... 16

Purification of Modified Peptide Synthesis...... 20

Removal of the Mmsb Amide Protecting Group...... 22

Desalting of Peptides...... 28

NMR spectra of Building Blocks...... 29

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Materials and General Methods

Amino acids used for peptide syntheses Fmoc-L-AA(PG)-OH were commercially available containing the common side chain protecting groups for Fmoc/tBu strategy. The solid supports (polystyrene and ChemMatrix), as well as the coupling reagents (DIPCDI, OxymaPure or HBTU), to synthesize the peptides were supplied from commercial vendors. Manual SPPS were carried out in polypropylene syringes fitted with a polyethylene porous disc; all synthetic cycles at room temperature, following the specified conditions described below.

Solvents (DMF and CH2Cl2) and soluble reagents were removed by suction. The automatic SPPS was done in a Microwave Peptide Synthesizer (Discover, CEM Corporation) with a software system Liberty and in a 0.1 mmol scale. The dipeptide amide formation to be used as building block was performed in a 10 mL sealed glass tube in a focused monomode microwave oven (Discover, CEM Corporation) featured with a surface sensor for internal temperature determination. Cooling was provided by compressed air, ventilating the microwave chamber during the reaction.

The HPLC characterizations were performed on a Waters instrument comprising a separation module (Waters 2695) and a photodiode array detector (Waters 2998) with a software system controller Empower. The analytical column used to characterize the synthesized building blocks and the major peptide sequences was a reverse-phase C18 column (XBridgeTM BEH130, 4.6 x 100 mm, 3.5 μm); except for Aβ(1-42) sequences that were analyzed in a reverse-phase C4 column (Symmetry300TM, 4.6 x 150 mm, 5 μm). UV detection was measured at 220 nm. Flow rate was at 1 mL/min. The eluent system used in all HPLC conditions was based in linear gradients of eluent B (MeCN +

0.036% TFA) into eluent A (H2O + 0.045% TFA). The HPLC gradients on a reverse-phase C18 column were performed over 8 min at 25 ºC; and on a reverse-phase C4 column were performed at 60ºC, over 60 min or 30 min, which is specified in every experiment.

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The HPLC purifications were carried out in two different Waters equipments: in the case of RADA-16, this peptide was purified with a semi-prep HPLC-MS (ESI) Waters 2545 binary gradient system comprising a sample manager (Waters 2767) and a UV detector (Waters 2489) with a dual absorbance selected at 254 and 220 nm. The column used was a reverse-phase C18 XBridgeTM BEH130, 419 x 100 mm, 5 μm OBDTM) where the conditions were

based in linear gradients of MeCN (+ 0.1% TFA) into H2O (+ 0.1% TFA). The MS module was a Micromass ZQ. The software used was a MassLynx version. In the case of Aβ(1-42) and also the dipeptide building block (Fmoc-Ala- N(Mmsb)-Ala-OH), a 600 Delta system comprising a sample manager (Waters 2747) and a UV detector (Waters 2487) with a dual absorbance selected at 254 and 220 nm was used. The software used was a MassLynx version. The columns used were specified in every case and the conditions were based in

linear gradients of MeCN (+ 0.1% TFA) into H2O (+ 0.1% TFA). The CombiFlash Rf 200 purification of certain intermediates of building block syntheses was performed in a Teledyne Isco system on a 50 g C18 RediSep Rf GOLD column. UV detector was measured at 254 nm with gradients of eluent B

(MeCN + 0.036% TFA) into eluent A (H2O + 0.045% TFA).

Circular Dichroism measurements were carried out in a Jasco J-815 CD spectrometer; at 25 ºC, and recorded from 250 to 190 nm at a scanning speed

of 50 nm/min and a time response of 4 s. Samples were dissolved in H2O/2,2,2- trifluoroethanol (TFE) (9:1) at 0.25 µM and deposited in a quartz cell of 1 mm path length. Each CD measurement was represented as an average of 4 repeated scans in steps of 0.2 nm. Data were processed with Jasco’s Spectra Manager Software and the measurements were converted from mdeg to molar ellipticities [] expressed in mdeg · cm2 · dmol-1.

Mass analyses were performed by different systems: the reverse phase-high performance liquid chromatography-electrospray mass spectrometry (RP- HPLC-ESI MS) analysis to characterize the building blocks was performed on a Waters Micromass ZQ spectrometer comprising a separation module (Waters 2695), an automatic injector (Waters 717 autosampler) and photodiode array

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detector (Waters 2998) with a software system controller MassLynx v. 4.1. UV detector was acquired at 220 nm and linear gradients of MeCN (+ 0.07% formic

acid) into H2O (+ 0.1% formic acid) at a flow rate of 0.3 mL/min over 8 min. The Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight mass spectrometry (MALDI-TOF MS) peptide characterizations were performed on an Applied Biosystems Voyager-DE RP instrument, using α-cyano-4-hydroxycinamic acid as a matrix. The characterization of impurity in unprotected Aβ(1-42) peptide was performed in a LC/MSD-TOF (Agilent Technologies) associated to a electrospray mass spectrometer (ESI-MS) and the sample was dissolved in

H2O/MeCN (1:1) containing 0.1% of formic acid. The HR-MS (ESI) spectrometry characterization of Aβ(1-42) peptide sequences were performed on a LTQ-FT Ultra (Thermo Scientific) spectrometer. The ionization was performed by a nanoESI (Advion BioSciences, Ithaca, NY, USA) with direct infusion of sample. The sample Mmsb protected Aβ(1-42) and the unprotected Aβ(1-42) were

dissolved in H2O/MeCN (1:1) with 0.5% of formic acid and were directly infused to MS analysis. Data were processed with Xcalibur software vs.2.0SR2 (ThermoScientific).

Desalting of the peptide was performed on a prepacked disposable PD MidiTrap™ column containing 5.3 ml of Sephadex™ G-10. These columns allow removing the salts because of different sizes compared with the sample (peptide) molecules larger are excluded from the Sephadex porous and eluted before the salts. The buffer chose to elute the peptide depends on the solubility of them. In the present work the RADA-16 peptide was eluted with 0.045% TFA

in H2O. The collected tubes were analyzed by HPLC and the fractions which contained the peptide sample were combined. Peptide sample eluted first and second the salts. Lyophilization gave the expected peptide without salts.

1H and 13C NMR spectra were recorded on a Varian MERCURY 400 (400 MHz for 1H NMR, 100 MHz for 13C NMR) spectrometer. Chemical shifts (δ) are expressed in parts per million downfield from tetramethylsilyl chloride. Coupling constants are expressed in Hertz.

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Building Block synthesis

A) Fmoc-N(Mmsb)-Ala-OH

3-methoxymethylthiobenzene (2)

In a round-bottomed flask, 3-methoxythiophenol (1) (2.50 mL,

20.2 mmol) was dissolved in CH2Cl2 (15.6 mL) and cooled in an ice-water bath and MeI (1.38 mL, 22.2 mmol) was then added. Then, triethylamine (3.09 mL, 22.2 mmol) was added dropwise and the mixture was warmed to room temperature and stirred for 30min. The course of the reaction was followed by HPLC. After

completion of the reaction, the organic phase was washed with H2O (3 x 10 mL) and

Brine (3 x 10 mL). The organic fractions were dried (MgSO4) and the solvent was removed in vacuo to afford the title compound 2 as a yellow oil, which was used in the next reaction without further purification (3.07 g, 99%).

HPLC analysis (tR = 4.09 min; gradient from 50% to 60% MeCN over 8 min). The main product corresponds to the expected title compound 2.

1 H NMR (400 MHz; CDCl3; Me4Si): δ 7.20 (t, J = 8.0 Hz, 1H), 6.85 (ddd, J = 7.8, 1.7, 0.8 Hz, 1H), 6.82 – 6.80 (m, 1H), 6.68 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H), 3.80

(s, 3H, OCH3), 2.48 (s, 3H, SCH3).

13 C NMR (100 MHz, CDCl3): δ 160.0 (C), 139.9 (C), 129.7 (CH), 118.9 (CH),

112.2 (CH), 110.7 (CH), 55.3 (OCH3), 15.8 (SCH3).

2-methoxy-4-methylthiobenzaldehyde (3)

A freshly Vilsmeier reagent (6 equiv) was prepared as follows: in a 250 mL round-bottomed flask, phosphorous oxychloride (10.68 mL, 116.7 mmol) was added followed by

dry CH2Cl2 (25 mL) under an argon atmosphere. The solution was cooled to 10 ºC and a solution of dry DMF (6.65 mL,

85.6 mmol) in dry CH2Cl2 (25 mL) was added dropwise. After the Vilsmeier reagent was prepared, a solution of 3-methoxymethylthiobenzene (2) (3 g, 19.5 mmol) in dry

CH2Cl2 (6 mL) was added slowly. The ice bath was removed and the solution was heated under reflux to 50 ºC and stirred for 15 h. After the reaction mixture had cooled 5

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to room temperature, the mixture was poured carefully into crushed ice and the product

was extracted with CH2Cl2 (3 x 25 mL). The organic layers were combined, dried

(MgSO4) and then, the solvent was evaporated under vacuum. Residual DMF was

removed by co-evaporation with toluene and CH2Cl2. The resulting pale brown solid contains as a major isomer the expected product in 55.8 %. This crude was subjected to purification by CombiFlash Rf 200 system (reverse phase C18 silica, linear gradient from (B/A) 5:95 to 30:70 over 40 min, then isocratic 30:70 over 40 min) to yield the title compound 3, after lyophilization, as a white solid (1.63 g, non optimized 48% yield).

HPLC analysis (tR = 2.80 min; gradient from 50% to 60% MeCN over 8 min). The expected product title compound 3 shows an HPLC-MS (ESI): m/z calcd for + C9H10O2S: 182.04, found: 183.21 [M + H] . The main impurity detected (HPLC

tR = 2.49 min; gradient from 50% to 60% MeCN over 8 min) corresponds to the other isomer (4-methoxy-2-methylthiobenzaldehyde), HPLC-MS (ESI): m/z + calcd for C9H10O2S: 182.04, found: 183.19 [M + H] .

1 H NMR (400 MHz; CDCl3; Me4Si): δ 10.33 (d, J = 0.6 Hz, 1H, CHO), 7.74 (d, J = 8.2 Hz, 1H), 6.83 (dd, J = 8.2, 1.1 Hz, 1H), 6.78 (d, J = 1.6 Hz, 1H), 3.91 (s,

3H, OCH3), 2.52 (s, 3H, SCH3).

13 C NMR (100 MHz, CDCl3): δ 188.9 (CHO), 161.8 (C), 149.6 (C), 129.0 (CH),

121.9 (C), 117.1 (CH), 108.1 (CH), 55.8 (OCH3), 14.9 (SCH3).

(S)-2-((2-methoxy-4-(methylthio)benzyl)amino)propanoic acid

[N(Mmtb)-Ala-OH] (4)

NaBH3CN (0.20 g, 3.1 mmol) was added to a suspension of 2-methoxy-4-methylthiobenzaldehyde (3) (0.50 g, 2.7

mmol,) and H-L-Ala-OH (0.19 g, 2.2 mmol) in H2O/dioxane (1:1) (6.6 mL). The pH was adjusted to 5-6 with 1 N HCl and the resulting solution was stirred overnight. The

solution was washed with CH2Cl2 (3 x 4 mL) and the aqueous layer was lyophilized to give the title crude 4 as a white solid (0.66 g), which was used in the next reaction without further purification.

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HPLC analysis (tR = 4.18 min; gradient from 5% to 100% MeCN over 8 min). The main product corresponds to the expected title compound 4, HPLC-MS + (ESI): m/z calcd for C12H17NO3S: 255.09, found: 256.00 [M + H] .

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-methoxy-4- (methylthio)benzyl)amino)propanoic acid

[Fmoc-N(Mmtb)-Ala-OH] (5) The lyophilized crude 2-methoxy-4-methylthiobenzyl alanine (4) was suspended in a mixture of 1%

aqueous Na2CO3/dioxane (1:1, v/v) (14 mL) and cooled with an ice bath. A mixture of Fmoc-OSu (0.75 g, 2.2 mmol) in dioxane (2.3 mL) was slowly added

keeping the pH at 9 with 10 % aq. Na2CO3. The reaction mixture was stirred overnight and the conversion was checked by HPLC. Extra Fmoc-OSu

(0.33 g) was required to complete the reaction. H2O (30 mL) was added to the reaction mixture, the pH was adjusted to 8 with 1 N HCl and washings with MTBE (3 x 20 mL) were performed. The aqueous phase was acidified to

pH 1 with HCl/H2O (1:2) and the product was extracted with EtOAc (3 x 30 mL). The

organic fractions were dried with MgSO4, concentrated under vacuo and the residue was purified using a CombiFlash Rf 200 system (reverse phase C18 silica, linear gradient from (B/A) from 0:100 to 50:50 over 50 min, then isocratic 50:50 over 10 min) to yield, after lyophilization, compound 5 as a white solid (0.43 g, 41% overall yield for two steps).

HPLC analysis (tR = 6.49 min; gradient from 50% to 80% MeCN for 8 min). The main product corresponds to the expected title compound 5, HPLC-MS (ESI): + m/z calcd for C27H27NO5S: 477.16, found: 478.02 [M + H] . The main impurity

detected (HPLC tR = 2.86 min; gradient from 50% to 80% MeCN over 8 min)

corresponds to Fmoc-L-Ala-OH, HPLC-MS (ESI): m/z calcd for C18H17NO4: 311.11, found: 312.02 [M + H]+.

1 H NMR (400 MHz, DMSO-d6; Me4Si) (mixture of rotamers): δ 7.92 – 7.63 (m, 3H), 7.47 – 7.30 (m, 4H), 7.20 (dt, J = 19.4, 7.4 Hz, 1H), 6.97 – 6.70 (m, 3H),

4.47 – 4.12 (m, 6H), 3.80 and 3.79 (s, 3H, OCH3), 2.49 and 2.48 (s, 3H, SCH3), 1.23 and 1.13 (d, J = 7.2 Hz, 3H). 7

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13 C NMR (100 MHz, DMSO-d6) (mixture of rotamers): δ 172.94 (COOH), 156.51 (CO from Fmoc), 155.45 (C), 143.70 and 143.61 (C), 140.67 (C), 137.75 and 137.59 (C), 127.88 (CH), 127.52 (CH), 126.95 and 126.91 (CH), 124.95 and

124.90 (CH), 123.14 (CH), 120.03 (C), 117.64 (CH), 108.38 (CH), 66.86 (CH2

from Fmoc), 55.59 (CH), 55.43 (CH3), 46.54 (CH), 44.74 (CH2), 14.88 (CH3),

14.81 (CH3).

(2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-methoxy-4- (methylsulfinyl)benzyl)amino)propanoic acid

[Fmoc-N(Mmsb)-Ala-OH] (6)

Acetic acid (12 mL) was added to a round bottomed- flask which contained 2-methoxy-4-methylthiobenzyl Fmoc-alanine (5) (0.38 g, 0.79 mmol). A solution of aqueous hydrogen peroxide (30%) (2.58 mL, 86.19 mmol) was slowly added to the reaction mixture while stirring, until total conversion was confirmed by HPLC. The mixture was poured onto crushed ice and the

product was extracted with CH2Cl2 (3 x 15 mL). The organic layers were combined and

dried (MgSO4). The organic solvent was evaporated in vacuo. Acetic acid was removed by co-evaporation with toluene, dichloromethane and diethyl ether to afford the compound Fmoc-N(Mmsb)-Ala-OH (6) as a white solid (0.43 g, 98.2% yield). This product was pure enough (98%) to be used on solid-phase peptide synthesis without further purification.

HPLC analysis (tR = 2.47 min; gradient from 50% to 80% MeCN for 8 min). The main product corresponds to the expected title compound 6, HPLC-MS (ESI):

m/z calcd for C27H27NO6S: 449.17, found: 449.07 [M].

1 H NMR (400 MHz, DMSO-d6; Me4Si) (mixture of rotamers): δ 7.94 – 7.63 (m, 3H), 7.39 (m, 4H), 7.27 (dd, J = 11.0, 4.7 Hz, 1H), 7.23 – 7.09 (m, 3H), 4.52 –

4.10 (m, 6H), 3.88 and 3.87 (s, 3H, OCH3), 2.76 (s, 3H, SOCH3), 1.26 and 1.15 (d, J = 7.2 Hz, 3H).

13 C NMR (100 MHz, DMSO-d6) (mixture of rotamers): δ 172.97 (COOH), 156.66 (CO from Fmoc), 155.53 (C), 145.93 (C), 143.64 and 143.58 (C), 140.67 (C),

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129.27 (CH), 127.65 (CH), 127.49 (CH), 126.90 and 126.85 (C), 124.82 and

124.75 (CH), 120.06 and 120.00 (CH), 115.27 (CH), 105.13 (CH), 66.84 (CH2

from Fmoc), 55.70 (CH3), 46.91 (CH), 46.54 (CH), 44.87 (CH2), 43.26 and 43.21

(CH3), 15.59 and 14.85 (CH3).

B) Fmoc-Ala-N(Mmsb)-Ala-OH

(2S)-2-((2-methoxy-4-(methylsulfinyl)benzyl)amino)propanoic acid

[H-N(Mmsb)-Ala-OH] (13)

Fmoc-N(Mmsb)-Ala-OH 6 (0.15 g, 0.3 mmol) was dissolved in CH2Cl2 (0.8 mL), and diethylamine (0.14 mL, 1.4 mmol) was added dropwise into the mixture with stirring. The reaction was monitored by HPLC until the reaction was completed (4-5 h). The solvent was removed in vacuo and the diethylamine was co-evaporated with toluene.

H2O was added and the dibenzofulvene adduct precipitated. The solid was removed by centrifugation and the aqueous phase was lyophilized to afford the compound H- N(Mmsb)-Ala-OH (13) as a white solid (0.07 g, 91.5%).

HPLC analysis (tR = 3.00 min; gradient from 5% to 50% MeCN for 8 min). The main product corresponds to the expected title compound 13, HPLC-MS (ESI): + m/z calcd for C12H17NO4S: 271.09, found: 272.08 [M + H] .

1 H NMR (400 MHz, CD3OD; Me4Si): δ 7.61 (d, J = 7.8 Hz, 1H), 7.44 (d, J = 1.4 Hz, 1H), 7.32 (dd, J = 7.8, 1.5 Hz, 1H), 4.28 (d, J = 3.0 Hz, 2H), 4.02 (s, 3H,

OCH3), 3.59 (q, J = 7.2 Hz, 1H), 2.82 (s, 3H, SOCH3), 1.53 (d, J = 7.2 Hz, 3H).

13 C NMR (100 MHz, CD3OD): δ 160.33 (COOH), 149.68 (C), 133.69 (C), 117.06

(CH), 117.04 (C), 107.04 (CH), 107.03 (CH), 56.73 (CH and CH3), 46.40 (CH2),

43.67 (CH3), 16.14 (CH3).

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(S)-(9H-fluoren-9-yl)methyl (1-fluoro-1-oxopropan-2-yl)carbamate

[Fmoc-L-Ala-F] (14)

In a round-bottomed flask, commercial Fmoc-L-Ala-OH (0.20 g, 0.6 mmol) was

dissolved in anhydrous CH2Cl2 (25 mL). The reaction mixture was cooled to 0ºC and stirred. The Bis(2-methoxyethyl)aminosulfur trifluoride (DeoxoFluor®) (0.17 mL, 0.8 mmol) was added dropwise into the mixture. After 45 min, extra DeoxoFluor® equivalents (0.17 mL, 0.8 mmol) were added to the reaction to complete the conversion. After a total of 1.75 h the reaction was stopped. The organic layer was

washed once with H2O (50 mL) and once with brine (50 mL). The organic phase was

dried (MgSO4) and the solvent was evaporated in vacuo to afford the compound Fmoc- L-Ala-F (14) as a brown solid (0.15 g, quantitative yield). The product was used in the next reaction without further purification.

HPLC analysis (tR = 4.61 min; gradient from 50% to 70% MeCN for 8 min). The main product corresponds to the expected title compound 14.

(2S)-2-((2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-methoxy- 4-(methylsulfinyl)benzyl)propanamido)propanoic acid

[Fmoc-Ala-N(Mmsb)-Ala-OH] (15)

Amide formation was carried out under MW conditions, in batches. H-N(Mmsb)-Ala-OH (0.03 g, 0.1 mmol) and Fmoc-L-Ala-F (0.02 g, 0.06 mmol) were placed in a microwave

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reactor vessel with dimethyl sulfoxide (2 mL) and N,N-diisopropylethylamine (DIEA) (0.01 mL, 0.06mmol). The mixture was heated under microwave irradiation at 35 ºC for 10 min. A total of 4 consecutive MW cycles were performed by adding Fmoc-L-Ala-F

(0.02 g, 0.06 mmol) in every cycle. H2O (4 mL) was added to the reaction mixture and

the product was extracted with CH2Cl2 (3 x 4 mL). The organic layers were combined,

dried (MgSO4) and the solvent was removed under reduced pressure. The compound Fmoc-Ala-N(Mmsb)-Ala-OH was obtained as an oil and subsequently purified [by reverse phase on a semi-prep HPLC with a XBridgeTM Prep C18 OBDTM (19 x 100, 5

m) column; the elution system was the following: B (MeCN + 0.1% TFA) into A (H2O + 0.1% TFA); linear gradient (B/A) at a flow rate of 16 mL/min: 40:60 isocratic over 20 min, then from 40:60 to 45:55 over 10 min and finally from 45:55 to 50:50 over 10 min. The product afforded was Fmoc-Ala-N(Mmsb)-Ala-OH (15) (0.012 g, 7.5%, overall yield for two steps). This purified product (94%, HPLC purity) was used directly in SPPS to

synthesize the H-(Ala)10-NH2 (8) by the dipeptide strategy.

HPLC analysis (tR = 3.27 min; gradient from 45% to 55% MeCN for 8 min). The expected product title compound 15 (24.8%, HPLC purity) was detected by + HPLC-MS (ESI): m/z calcd for C30H32N2O7S: 564.19, found: 565.22 [M + H] .

1 H NMR (400 MHz; DMSO-d6; Me4Si) (mixture of rotamers): δ 7.89 (d, J = 7.5 Hz, 1H), 7.76 – 7.55 (m, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.35-7.14 (m, 3H), 4.93 – 4.62 (m, 1H), 4.60-4.50 (m, 2H), 4.33 – 4.16 (m, 4H), 3.89 and 3.88 (s, 3H,

OCH3), 2.73 and 2.71 (s, 3H, SOCH3), 1.21 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 8.9 Hz, 3H).

13 C NMR (100 MHz, DMSO-d6) (mixture of rotamers): δ 173.69 (COOH), 173.16 (CON), 157.27 (C), 155.92 (CO from Fmoc), 146.91 (C), 144.40 and 144.31 and 144.18 (C), 141.13 (C), 128.81 and 128.75 (CH), 128.06 (CH), 127.50 (CH), 125.83 (CH), 125.78 (C), 120.51 (CH), 115.75 (CH), 105.67 and 105.62 (CH),

66.08 (CH2 from Fmoc), 56.26 (CH3), 47.53 (CH), 47.07 (CH), 47.02 (CH),

43.68 (CH3), 43.53 (CH2), 18.18 and 18.16 (CH3), 14.72 (CH3).

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Solid-Phase Peptide Synthesis

Synthesis of Modified Peptide Sequences

a) Synthesis of Modified RADA-16

Incorporation of the incoming Fmoc-AA-OH onto H-N(Mmsb)-Ala-OH

In the synthesis of modified RADA-16 (11), an accurate coupling optimization of the incoming amino acid [Fmoc-Asp(tBu)-OH] was performed and summarized in a table (Table S1). The best coupling conditions found were: Fmoc-Asp(tBu)- OH (10 equiv) with the coupling reagent system DIPCDI (10 equiv) and OxymaPure (10 equiv) at 45ºC for 2 h. A total of 3 consecutive couplings (using the same coupling conditions) were carried out (Table S1, entry 10). The extension of this reaction was evaluated by HPLC analysis of the peptide crude

after an aliquot of peptidyl resin was treated with TFA/TIS/H2O (95:2.5:2.5) for 1 h. The solvent was evaporated and the peptide was precipitated with cold diethyl ether.

a HPLC method, analyzed after cleavage of the peptide from the resin. b OxymaPure: Ethyl 2-cyano-2- (hydroxyimino)acetate. c Bis(trichloromethyl) carbonate. d 2,4,6-trimethylpyridine. e Two consecutive cycles.

Table S1. Coupling conditions to introduce the incoming amino acid (Fmoc-L-Asp(tBu)-OH) to H-N(Mmsb)-Ala-Arg-Ala-Asp-Ala-Rink CM resin in the synthesis of modified RADA-16 (11).

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In the syntheses of modified H-(Ala)10-NH2 (8 and 9), the conditions used to couple the incoming amino acid (Fmoc-L-Ala-OH) were the described in entry 10 (Table S1).

b) Synthesis of Modified H-(Ala)10-NH2 (8) by Dipeptide Strategy

The sequence was synthesized on Rink-Amide AM Polystyrene resin (25 mg, 0.01 mmol, 0.45 mmol/g). The resin was conditioned by washings with DMF (3

x 1 min) and CH2Cl2 (3 x 1 min). Commercial Fmoc-L-Ala-OH was coupled as follows: Fmoc-L-Ala-OH (3 equiv), DIPCDI (3 equiv) and OxymaPure (3 equiv) in DMF, with a 5-min preactivation and a total coupling time of 1 h. After every

coupling the resin was washed with DMF (3 x 1 min) and CH2Cl2 (3 x 1 min). Then, a Kaiser test was carried out to check which amino acid was not completely coupled (in this case no uncompleted couplings were detected). No double couplings were carried out.

Incorporation of the building block Fmoc-Ala-N(Mmsb)-Ala-OH

After Fmoc removal of the previous amino acid, the purified Fmoc-Ala- N(Mmsb)-Ala-OH was coupled as follows: Fmoc-Ala-N(Mmsb)-Ala-OH (1.5 equiv), benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) (1.5 equiv) and DIEA (3 equiv) in DMF for 1.5 h.

Then, washings with DMF and CH2Cl2 were performed and a Kaiser test confirmed the complete incorporation of the AA.

Cleavage/Global Deprotection of Side Chain Protecting Groups

After the peptide elongation of the modified sequence, the cleavage and post- cleavage treatments were performed following the same conditions described

as for the corresponding standard and the two modified H-(Ala)10-NH2 sequences. The peptide crude was analyzed by HPLC and MALDI-TOF MS.

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Analysis of Peptide Crudes

A) Analysis of Standard Peptide Sequences

a) H-(Ala)10-NH2 Standard (7): Sample preparation: 1 mg of peptide crude

H-(Ala)10-NH2 (7) was completely dissolved in 10μL of TFA after sonication.

Then, 1 mL of H2O/MeCN (1:1) was added, although no completed dissolved

peptide crude was obtained. HPLC analysis (Figure S1a) (tR = 3.72 min; gradient from 5% to 40% MeCN over 8 min). The main product (66%, HPLC

purity) corresponds to the expected standard H-(Ala)10-NH2 (7), MS (MALDI- + TOF) (Figure S1b): m/z calcd for C30H53N11O10: 727.40, found: 728.60 [M + H] , 750.50 [M + Na]+ and 766.60 [M + K]+. Impurities associated with Ala deletions: + H-(Ala)9-NH2 m/z Calcd for C27H48N10O9: 656.36, found: 657.60 [M + H] and + 679.60 [M + Na] ; H-(Ala)8-NH2 m/z Calcd for C24H43N9O8: 585.32, found: + + 586.50 [M + H] and 608.50 [M + Na] ; H-(Ala)7-NH2 m/z Calcd for C21H38N8O7: + + 514.29, found: 515.40 [M + H] and 537.4 [M + Na] ; H-(Ala)6-NH2 m/z Calcd for + + C18H33N7O6: 443.25, found: 444.40 [M + H] and 466.40 [M + Na] ).

Figure S1. Synthesized Standard H-(Ala)10-NH2 (7): HPLC chromatogram (a); and MALDI-TOF MS spectrum (b).

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b) RADA-16 Standard (10): Sample preparation: 1 mg of peptide crude RADA- 16 (10) was completely dissolved in 10μL of TFA after sonication. Then, 1 mL of

H2O/MeCN (1:1) was added, being these the best conditions to dissolve the

RADA-16. HPLC analysis (tR = 5.00 min; gradient from 5% to 25% MeCN for 8 min). The main product (88%, HPLC purity) corresponds to the expected standard RADA-16 (10), MS (MALDI-TOF) (Figure S2): m/z calcd for + C66H113N29O25: 1711.85, found: 1712.90 [M + H] . Impurities were also detected, such as the peptide sequence with one Ala deletion m/z calcd for + C63H108N28O24: 1640.81, found: 1663.2 [M + Na] ; the peptide sequence with + one Arg deletion m/z calcd for C60H101N25O24: 1555.75, found: 1556.9 [M + H] ;

the peptide sequence with two Ala deletions m/z calcd for C58H101N27O22: + 1527.76, found: 1550.7 [M + Na] ; Ac-(RADA)3-NH2 m/z calcd for C50H86N22O19: + 1298.64, found: 1299.7 [M + H] ; H-(RADA)3-NH2 m/z calcd for C48H84N22O18: 1256.63, found: 1257.7 [M + H]+ and the 13-mer peptide m/z calcd for + C51H89N23O19: 1327.67, found: 1328.7 [M + H] .

Figure S2. MALDI-TOF MS spectra of fractions collected from HPLC analysis of Standard RADA-16 (10).

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B) Analysis of Modified Peptide Sequences

a) Modified H-(Ala)10-NH2 in position 6 (8): Sample preparation: 1 mg of

peptide crude (8) was completely dissolved in 1 mL of H2O/MeCN (1:1). HPLC

analysis (Figure S3) (tR = 4.00 min; gradient from 5% to 60% MeCN over 8 min). The main product (74%, HPLC purity) corresponds to the expected modified H-

(Ala)10-NH2 (8), MS (MALDI-TOF): m/z calcd for C39H63N11O12S: 909.44, found: + 932.60 [M + Na] . The product that eluted at tR = 4.89 min (5%, HPLC purity) corresponds to the expected sequence but with the sulfoxide oxidized to sulfone + MS (MALDI-TOF): m/z calcd for C39H63N11O13S: 925.43, found: 948.6 [M + Na]

and the byproduct that eluted at tR = 3.18 min (9%, HPLC purity) corresponds to

the truncated sequence 5-mer, MS (MALDI-TOF): m/z calcd for C24H38N6O7S: 554.25, found: 555.29 [M + H]+.

Figure S3. HPLC chromatogram of the modified H-(Ala)10-NH2 crude peptide (8).

b) Modified H-(Ala)10-NH2 in position 8 (9): Sample preparation: 1 mg of

peptide crude (9) was completely dissolved in 1 mL of H2O/MeCN (1:1). HPLC

analysis (Figure S4) (tR = 3.96 min; gradient from 5% to 60% MeCN over 8 min). The main product (63%, HPLC purity) corresponds to the expected modified H-

(Ala)10-NH2 (9), MS (MALDI-TOF): m/z calcd for C39H63N11O12S: 909.44, found: + 932.45 [M + Na] . The peak that eluted at tR = 5.01 min (2%, HPLC purity) corresponds to the expected sequence with the sulfoxide oxidized

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to sulfone, MS (MALDI-TOF): m/z calcd for C39H63N11O13S: 925.43, found: + 948.50 [M + Na] and the byproduct that eluted at tR = 3.06 min (6%, HPLC purity) corresponds to the truncated sequence 3-mer, MS (MALDI-TOF): m/z + calcd for C18H28N4O5S: 412.18, found: 413.30 [M + H] .

Figure S4. Modified H-(Ala)10-NH2 (9) crude analysis by: HPLC (a) and MALDI-TOF MS (b).

c) Modified RADA-16 (11): Sample preparation: 1 mg of peptide crude (11)

was completely dissolved in 1 mL of H2O/MeCN (1:1). HPLC analysis (Figure

S5a) (tR = 5.91 min; gradient from 5% to 25% MeCN over 8 min). The main product (89%, HPLC purity) corresponds to the expected modified RADA-16

(11), MS (MALDI-TOF) (Figure S5b): m/z calcd for C75H123N29O27S: 1893.89, + found: 1895.17 [M + H] . The peak that eluted at tR = 6.46 min (3.6%, HPLC purity) corresponds to the expected sequence with the sulfoxide oxidized to

sulfone, MS (MALDI-TOF): m/z calcd for C75H123N29O28S: 1909.88, found + 1911.1 [M + H] and the byproduct that eluted at tR = 3.88 min (0.7%, HPLC

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purity) corresponds to the truncated sequence 5-mer, MS (MALDI-TOF): m/z + calcd for C28H45N9O9S: 683.31, found: 684.4 [M + H] .

Figure S5. Modified RADA-16 (11) crude analysis by: HPLC (a) and MALDI-TOF MS (b).

d) Modified Aβ(1-42) (12): Sample preparation: 1 mg of crude peptide (12) was completely dissolved in 1 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP).

HPLC analysis (tR = 19.28 min; C4 column at 60 ºC, gradient from 10% to 50% MeCN for 30 min). The main product (89%, HPLC purity) corresponds to the expected modified Aβ(1-42) (12), MS (MALDI-TOF) (Figure S6): m/z calcd for + C212H321N55O62S2: 4693.31, found: 4714.94 [M + Na] . In order to obtain more exact measurements by mass spectrometry, an accurate HR-MS (ESI) (Figure S7) analysis was performed, confirming the detection of the expected peptide: + m/z calcd for C212H321N55O62S2: 4693.3098, found: 1129.3273 [M + 4H] /4, 903.6643 [M + 5H]+/5 and 753.3872 [M + 6H]+/6.

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Figure S6. MALDI-TOF MS spectrum of modified Aβ(1-42) (12).

Figure S7. HR-MS (ESI) spectrum of modified Aβ(1-42) (12).

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e) Modified H-(Ala)10-NH2 (8, by dipeptide strategy): Sample preparation: 1

mg of peptide crude was completely dissolved in 1 mL H2O/MeCN (1:1). HPLC

analysis (Figure S8) (tR = 3.81 min; gradient from 10% to 40% MeCN for 8 min). The main product (97%, HPLC purity) corresponds to the expected modified H-

(Ala)10-NH2 (8), MS (MALDI-TOF): m/z calcd for C39H63N11O12S: 909.44, found: 932.45 [M + Na]+. The peak corresponding to the truncated peptide (5 mer) was not detected.

Figure S8. Analysis of H-(Ala)10-NH2 8 by Dipeptide strategy by: HPLC (a) and MALDI-TOF MS (b).

Purification of Modified Peptides

A) Modified RADA-16 (11): 65 mg of modified RADA-16 crude were divided in

4 batches (approx. 15 mg each). Every portion was dissolved in H2O/MeCN (1:1) (0.9 mL) and injected into a semi-preparative HPLC provided with a reverse-phase C18 column (XBridgeTM Prep BEH130, Waters: 19 x 100 mm, 5

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m). A linear gradient of (%B) 5-10% over 1 min followed by 10-20% over 7 min was followed, at a flow rate of 16 mL/min: The elution system was the following:

B (MeCN + 0.1% TFA) into A (H2O + 0.1% TFA). After purification and lyophilization, 27.1 mg of pure modified RADA-16 were obtained with a 41.7% purification yield, and excellent purity (99.9%). HPLC analysis of purified

product (tR = 5.116 min; gradient from 5% to 25% MeCN for 8 min) and the MS (MALDI-TOF) confirmed the mass of the expected product (Figure S9): m/z + calcd for C75H123N29O27S: 1893.89, found: 1894.69 [M + H] .

Figure S9. MALDI-TOF MS spectrum of pure modified RADA-16 (11).

B) Modified Aβ(1-42) (12): Some portion of A(1-42) was dissolved in HFIP (0.8 mL) and injected in a semi-preparative HPLC provided with a reverse- phase C4 column (Jupiter, Phenomenex: 21 x 150, 10m). A linear gradient of two eluents and flow rate of 20 mL/min (%B): 0-10% in 5 min followed by 10- 50% in 60 min was followed. The elution system followed: B (MeCN + 0.1%

TFA) into A (H2O + 0.1% TFA). After purification and lyophilization, modified Aβ(1-42) was obtained with 90% of purity (measured by HPLC). HPLC analysis

of purified product (tR = 18.492 min; C4 column at 60ºC, gradient from 10% to 50% MeCN for 30 min) and the MS (MALDI-TOF) confirmed the mass of the

expected product: m/z calcd for C212H321N55O62S2: 4693.31, found: 4714.19 [M + Na]+ and 2360.98 [M + 2H]+/2. In order to obtain more exacted measurements by mass spectrometry, an accurate HR-MS (ESI) analysis was performed, confirming the detection of the expected peptide: m/z calcd for

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+ C212H321N55O62S2: 4693.3098, found: 1174.8373 [M + 4H] /4 and 940.0719 [M + 5H]+/5.

Figure S10. MALDI-TOF MS spectrum of pure modified Aβ(1-42) (12).

Figure S11. HR-MS (ESI) spectrum of pure modified Aβ(1-42) (12).

Removal of the Mmsb Amide Protecting Group

A) Modified H-(Ala)10-NH2 at Position 6 (8)

2 mg of pure modified H-(Ala)10-NH2 (8) was dissolved in neat TFA (2 mL), and ammonium iodine (3.6 mg) was added to the mixture. The reaction was monitored by MALDI-TOF MS and was stopped after 2 h. The work-up was accomplished by removing the solvent by evaporation and further precipitation and washings of the peptide with cold diethyl ether to afford the expected unprotected product as a white solid peptide. Analysis by MS (MALDI-TOF)

revealed the completed reaction: m/z calcd for C30H53N11O10: 727.40, found: + + 750.42 [M + Na] and 766.39 [M + K] . The starting material [modified H-(Ala)10-

NH2 (8)] was not detected.

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B) Modified H-(Ala)10-NH2 at Position 8 (9)

2 mg of pure modified H-(Ala)10-NH2 (9) was dissolved in neat TFA (2 mL), ammonium iodine (3.6 mg) was added to the mixture. The reaction was monitored by MALDI-TOF MS (Figure S12) and it was stopped after 2 h. The work-up was accomplished by removing the solvent by evaporation and precipitation and washings with cold diethyl ether to afford the expected unprotected product as a white solid peptide. MS (MALDI-TOF) analysis

revealed the completed reaction: m/z calcd for C30H53N11O10: 727.40, found: + + 750.43 [M + Na] and 766.41 [M + K] . The starting material [modified H-(Ala)10-

NH2 (9)] was not detected.

Figure S12. MALDI-TOF MS spectra of monitoring Mmsb removal of modified H-(Ala)10-NH2 at

position 8 (9). Starting material (Mmsb protected H-(Ala)10-NH2 (9)) (blue). Final product

(unprotected H-(Ala)10-NH2) (green). 23

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C) Modified RADA-16 (11)

5 mg of pure modified RADA-16 (11) were dissolved in neat TFA (5 mL), and ammonium iodine (11.5 mg) was added to the mixture. The reaction was monitored by MALDI-TOF MS (Figure S13) and it was stopped after 45 min. The work-up was accomplished by removing the solvent by evaporation, precipitation and washings with cold diethyl ether to afford the expected unprotected product as a white solid peptide. MS (MALDI-TOF) analysis

revealed the completed reaction (Figure S14): m/z calcd for C66H113N29O25: 1711.85, found: 1713.00 [M + H]+ and 856.97 [M + 2H]+/2. The starting material [RADA-16 (11) was not detected.

Figure S13. MALDI-TOF MS spectra monitoring of Mmsb removal from modified RADA-16 (11). Intermediate of the reaction (Mmsb protected RADA-16) (orange). Final product (unprotected RADA-16) (green).

Figure S14. MALDI-TOF MS spectrum of final lyophilized unprotected RADA-16 after Mmsb removal.

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D) Modified Aβ(1-42) (12)

0.8 mg of pure modified Aβ(1-42) were dissolved in neat TFA (800 L), and ammonium iodine (1 mg) was added to the mixture. The reaction was stopped after 2 h. The work-up was accomplished removing the solvent by evaporation, precipitation and washings of the peptide with cold diethyl ether and the characterization showed the expected unprotected product as a white solid in 90% purity. MS (MALDI-TOF) analysis revealed the completed reaction (Figure + S15): m/z calcd for C203H311N55O60S: 4511.27, found: 4714.20 [M + Na] and 2360.98 [M + 2H]+/2. The starting material [modified A(1-42) (12)] was not detected. In order to obtain more exact measurements by mass spectrometry, an accurate HR-MS (ESI) analysis (Figure S16) was performed, confirming the

detection of the expected peptide: m/z calcd for C203H311N55O60S: 4511.2696, found: 1129.3273 [M + 4H]+/4, 903.6643 [M + 5H]+/5 and 753.3872 [M + 6H]+/6.

Figure S15. MALDI-TOF MS spectrum of final lyophilized unprotected Aβ(1-42) after Mmsb removal.

Figure S16. HR-MS (ESI) spectrum of final lyophilized unprotected Aβ(1-42) after Mmsb removal.

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The 10% impurity detected by HPLC (tR = 20.3 min; C4 column at 60ºC, gradient from 10% to 50% MeCN for 30 min) was isolated and characterized by mass spectrometry ESI-MS. The UV profile (Figure S17) indicates the presence of the synthesized protecting group and the mass value confirms that the impurity corresponds to the truncated peptide protected in N-terminal with the sulfoxide reduced (Mmtb-21Ala-1Ala-OH, Figure S18).

Figure S17. HPLC photodiode array analysis of final lyophilized unprotected Aβ(1-42) after Mmsb removal including the UV profile of every product detected.

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Figure S18. ESI-MS spectra of isolated impurity from lyophilized unprotected Aβ(1-42) after

Mmsb removal (tR = 20.3 min, Figure S17). 27

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Desalting of Peptides

Modified RADA-16 (11): The RADA-16 crude obtained after Mmsb-removal

(1.6 mg) was dissolved in 1% TFA and H2O/MeCN (1:1) (1.6 mL) and the solution was passed through a Sephadex G-10 column. The buffer chosen for

the elution of the peptide was 0.045% TFA in H2O. After filling up the column with a total of 2000 L of buffer fractions were checked by HPLC, and those containing peptide were lyophilized to remove the liquid to afford the desalted peptide.

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NMR spectra of Building Blocks

1 [Fmoc-N(Mmtb)-Ala-OH] (5) H NMR (400 MHz, DMSO-d6; Me4Si)

13 C NMR (100 MHz, DMSO-d6)

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1 [Fmoc-N(Mmsb)-Ala-OH] (6) H NMR (400 MHz, DMSO-d6; Me4Si)

13 C NMR (100 MHz, DMSO-d6)

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1 [H-N(Mmsb)-Ala-OH] (13) H NMR (400 MHz, CD3OD; Me4Si)

13 C NMR (100 MHz, CD3OD)

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1 [Fmoc-Ala-N(Mmsb)-Ala-OH] (15) H NMR (400 MHz, DMSO-d6; Me4Si)

13 C NMR (100 MHz, DMSO-d6)

32

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CHAPTER 3. Peptide-Based Solubilizing Tag Strategies

Introduction

CHAPTER 3: Peptide-Based Solubilizing Tag Strategies Introduction

1. Enhancement of Polarity by Peptide-Based Structures

Short peptide sequences may be used as fragment units to be attached to other molecules/peptides in order to induce physico-chemical changes. These peptides, which act as properties modulators, are named tags and the main purpose described regarding their function is enhancing the polarity or solubility of a certain molecule. Homooligo-peptides are the most studied short peptide tags, specifically those which comprise charged side-chains, such as the basic Lys/Arg and the acidic Glu/Asp.

The incorporation of ionic short peptide-based solubilizing tags into a non-polar peptide through the C-terminus has been reported for several insoluble sequences. As it was aforementioned in the general introduction section, when the tag and the non•polar molecule are conjugated permanently (for example through an amide bond), after the cleavage, the peptide-tag complex is isolated and used directly for the desired purpose. Examples of nanoparticles containing peptide-based solubilizing tags have been described as drug carriers or metal chelators, among others, because of their biocompatibility. On the other hand, when the tag is conjugated temporarily to the peptide through a bifunctional spacer (linker), after the detachment from the resin, the peptide-tag remains attached due to the stability of the spacer under the cleavage conditions. The isolated peptide-tag conjugate has its physico-chemical properties modified temporarily, facilitating not only its manipulation in solution in terms of chromatographic/spectrometric characterization, but also its purification. Finally, the short peptide tag is detached from the non-polar peptide by a single chemical treatment, rendering the native peptide sequence. When the tag, together with the linker, acts as a semipermanent group, it may be considered a C-terminal protecting group, which temporarily protects the carboxylic function and, in parallel, contributes to enhance the solubility.

1.1. Ionic Short Peptide-Based Solubilizing Tags

Principally, charged AAs (Glu, Asp, Lys, and Arg) are those selected to be conjugated and enhance the solubility of non-polar molecules. Most reported studies have focused on the synthesis of proteins, based on three principal applications: (i) drug delivery, (ii) structured material-based and (iii) as solubilizing tool. The biocompatibility of drug delivery systems is mandatory and conjugates that contain some of these charged AAs fit in this requirement. These anionic poly-AA comprising Glu (also named PGA) or Asp (also named PAA) have been designed to be used as drug carrier systems, principally, synthesized by polymerization with PEG1,2 (Fig. 1a). The importance of their application

149 CHAPTER 3: Peptide-Based Solubilizing Tag Strategies Introduction

in medicine has become evident specially in those cases where an existent drug has demonstrated an increased efficiency against cancer when it is carried by a PEG-PolyAA complex.3–5 These two drug delivery systems take advantage of its pH sensitivity associated to the poly-AA component, which allow the design of pH-dependent release systems.6–8 Therefore, the drug is led through the biological system and specifically delivered to those tissues where the pH causes a chemical modification to the tag.

Regarding the second mentioned application, the micelles formation may be either composed by copolymers in conjugation with pegylated systems6,9,10 (Fig. 1b), or in alternation with other AAs.11,12 Moreover, other interesting materials composed by conjugating PEG, in this case to a poly-Arg, were studied as biodegradable hydrogels capable to encapsulate and release certain drugs.13

Finally, the subject of the present work, the connection of poly-AA to non-polar molecules to enhance its solubility in solution has been studied in different fields, such as the solubilization of: extremely non-polar organic compounds (graphene by poly•Lys);14 or inorganic cations (calcium trough poly-Glu),15 or even drugs with clear poor solubility in aqueous systems (by poly-Glu).16 This last application would be directly associated to facilitating its characterization or purification, thereby large molecules, such as proteins conjugated permanent- or temporarily to these solubilizing poly-AA, become fruitfully manipulable. Different articles have reported interesting results about protein solubilization by poly-Asp,17 poly-Glu,18 poly-Arg,19,20 or poly•Lys.21

a) b)

PEG-polyAA

drug

Figure 1. Two ionic peptide-based solubilizing structures formed by: (a) PEG-poly-Arg conjugate example; and (b) schematic representation of one micelle formed by the copolymer PEG and poly-AA with a conjugated drug on the surface and inside the core.

The spatial arrangement of AA side-chains into a branched morphology plays a significant role in the polarity, more than those that are exposed as linear primary structures. Short branched peptides, combined in some cases with PEG moieties, are easily synthesized and, depending on the charged AA selected as the branched

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function, they are able to be used in biological systems, such as: cell internalization in case of the Arg-based tag;22 drug carriers in case of the Glu-based tag23 (Fig. 2a) or even bone regeneration in case of the Asp-based tag.24 The charged lysine is one of the preferred AAs to form the core of branched structures because of the amino groups content, its ε-amino side-chain and in its α-amino, which allow the double amide bond formation per AA2 (Fig. 2c). These highly branched structures are named in some cases dendrimers and, when peptide components are present in the structure, they are sub•classified as the peptide dendrimers family (Fig. 2b). Principal dendrimer characteristics rely on their globular structure, which allow them to functionalize the branches offering the maximum functional groups exposed in a minimum surface, feature that makes them suitable as drug carriers. Peptides integrated as dendrimer components confer to the system water-solubility, biodegradability and high branched versatility to form globular structures. Although the first, and most used dendrimer, is the known as PAMAM (formed by poly(amidoamine), it does not contain a peptide component, several publications reveal the successful improvements when peptide dendrimers are used as drug delivery systems.25,26

a) (COOH)n (COOH)n

NH2 (Glu)n (Glu)n NH2

NH2 (Glu) NH n (Glu)n 2

(COOH)n (COOH)n

b) Arg c) drug NH2 Arg Lys Arg NH2 NH Lys Arg 2 Lys Arg NH2 Lys NH2 Lys Arg NH2 Lys Arg NH2 Lys Arg NH2

Figure 2. Three schematic representations of ionic peptide-based solubilizing structures composed of: (a) branched poly-Glu system; (b) dendrimer guanidinium functionalized, attributed to Arg; and (c) Lys-core branched structure conjugated with drug.

On the other hand, when linear or branched poly-peptides are designed to be temporarily conjugated to a non-soluble molecule, modulation of properties attributed to the connected tag is profitable basically to characterize or purify the target molecule. In this sense, although solubilizing proteins are well reported in the literature (previously mentioned in the general introduction section), only a few articles may be found regarding the solubility enhancement of non-polar peptides. In 1996, Englebretsen27 and co-workers proposed, for the Boc/Bzl strategy, the SPPS of

(Gly•Arg)4 sequence as solubilizing tag connected to the hydrophobic dodecaalanine

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peptide through the basic-labile hydroxyacetic acid linker. After cleaving the peptide from the resin by HF, the peptide remains connected to the sequence, being the purification a feasible step. Finally, a last treatment with triethylamine renders the dodecaalanine sequence. Later, in 1998, the same author proposed,28 for the Fmoc/tBu methodology, the basic-labile 4-hydroxymethylbenzoic acid (HMBA) linker for the identical purpose, this time with a series of Gly(Lys/Arg-Gly1-2)3-4-6 tags. Again, after cleavage from the resin, the peptide remains connected to the solubilizing tag allowing its purification, and subsequently, under 0.1 M NaOH, the tag is hydrolyzed yielding the desired peptide. Later, in 2009, two parallel works developed by Wade29 and Brimble,30 by applying the strategy described in 1998, used the same HMBA linker to conjugate a non-soluble peptide to the solubilizing tags: (Arg)6 or (Lys)5, respectively. One of the limitations associated to these linkers is the fact that cleavages are performed under basic conditions, a media known because of favoring the aspartimide side-reaction. 31 More recently, in 2014, a (Lys)4 tag connected to a non-polar peptide through the acid-labile 4-(hydroxymethyl)phenylacetic linker was described to be compatible only with the Fmoc/tBu strategy, but with the advantage of being totally free of aspartimide formation.

2. C-Terminal Protecting Groups

When peptides are synthesized on solid-phase by standard sequential AA incorporations, carboxylic acid protecting groups are not required, since the α-carboxylic acid from the incoming AA is the only carboxylic moiety susceptible to react with the α-amino group from the peptidyl-resin. Moreover, when the AA is coupled to the resin, or to the peptidyl-resin, its carboxylic group becomes protected, thus the solid support acts itself as carboxylic acid protector. However, there are some specific cases where the use of a carboxylic acid protection is mandatory: (i) on-resin "head-to-tail" cyclizations (Fig. 3b), (ii) attachment of the AA to the resin mediated by its side-chain (Fig. 3a), and (iii) in the reverse N→C peptide synthesis (Fig. 3c).

On the other hand, when peptides are synthesized in solution, the amide bond formation entails the presence of carboxylic protectors, because the absence of resin promotes competition between the carboxylic acid from the incoming AA and those from the other fragment. Thereby, strategies that combine solid-phase peptide methods and solution approaches, such as enzymatic-catalyzed peptide synthesis32 or peptide fragment condensation in solution, demand a wide range of carboxylic acid protecting groups. Specifically, those carboxylic acid protectors comprised in the C-terminus of a peptide fragment, are known as C-terminal protecting groups.

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PG1 O a) H N Ct-PG 2 H2N OH cleavage PG2 PG2 Ct-PG b) PG removal

PG1 O PG1 PG1 c) H2N OH PG reverse N→C PG2 PG 2 coupling

Figure 3. Solid-phase peptide synthesis situations when C-terminal protecting group is required. (a) Side- chain anchoring; (b) on solid-phase "head-to-tail" cyclization; and (c) reverse N→C peptide synthesis.

Some researchers have focused their attention on modifying the C-terminal function permanently33–35 or temporarily.36,37 Otherwise, several authors have centered their research on protecting the C-terminal of peptides when the resin does not act as a protector. Since peptide fragment condensation was initially described to reach large peptides, a broad range of C-terminal protectors emerged in the literature.38,39 Obviously all described C-terminal protectors are suitable for carboxylic acid side•chains of AAs, such as Asp or Glu. These protectors, depending on the conditions which allow their removal, may be classified in acid-labile, basic-labile or other lability conditions. Based on these parameters, some of the most used carboxylic acid protecting groups are exposed below.

2.1. Removable Under Acidic Conditions

The C-terminal protecting groups removable under acidic conditions may be used in the Fmoc/tBu SPPS strategy, since this carbamate is removed under basic conditions, and are also orthogonal to the Alloc moiety because of its lability to palladium conditions. The most used C-terminal protector belonging to the acid-labile group is the tert-butyl, tBu (removed under 90% TFA in DCM, or 4 M HCl in dioxane) (Fig. 4).

tBu R , R , R = OMe or H 1 2 3 Dmb Phenyl-EDOTn 2-CT

derivatives

Figure 4. The most significant described C-terminal protecting groups removable under acidic conditions.

Although there are other acid-labile protectors removable under equivalent acidic proportion, in this section are highlighted those labile to low acidic conditions. These

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groups permit the use of acid-resistant resins because, whereas the resin remains stable, these carboxylic protectors may be removed. An example of those is the 2,4•dimethoxybenzyl, Dmb40 (removed under 1% TFA in DCM) (Fig. 4). Some reports described the preparation of this kind of groups, as well as the wide application of them in peptide syntheses, demonstrating to be suitable groups to assemble peptide fragments in solution.32,33,41 Similarly, it is found the 2-chlorotrityl, 2-CT42,43 (removed under 1% TFA in DCM) (Fig. 4) and the 5-phenyl-3,4-ethylenedioxythenyl derivatives, phenyl-EDOTn44 (removed under 0.01-0.5% TFA in DCM) (Fig. 4), described as a very acid-sensitive protecting group.

2.2. Removable Under Basic Conditions

The basic labile C-terminal protecting groups may be suitable for the Boc/Bzl SPPS strategy, since this carbamate is removed under acidic conditions, and these protectors are also orthogonal to the Alloc moiety. Principal basic-labile C-terminal protectors found in the literature are the methyl or ethyl, Me, Et (removed under NaOH, KOH, or LiOH) (Fig. 5) which are used on solid-phase, but also in solution to perform peptide fragment condensations.32,45,46 The 9-fluorenylmethyl, Fm47,48 (removed under 15% DIEA in DMF, or 20% piperidine in DMF or DCM) (Fig. 5) described also in those cases where the carboxylic moiety from AA side-chain is orthogonally protected by other 49,50 51,52 groups, or the carbamoylmethyl, Cam (removed under 0.5 M NaOH, or Na2CO3 in H2O/DMF) (Fig. 5). Specifically, Cam protector was exploited in enzymatic-catalyzed peptide synthesis,53 and due to its biocompatibility with enzymes, together with its acyl-donating capacity, has become one of the preferred C-terminal protector for this kind of chemistry. Moreover, this protector has been used during the last decade contributing to the successful completion of large peptides.54–56 Another significant basic-labile C-terminal protector is the 4-{N-[1-(4,4-dimethyl-2,6- dioxocyclohexylidene)-3-methylbutyllamino}benzyl, Dmab57 [removed under hydrazine in H2O/DMF (2:98)] (Fig. 5) and, although some authors have reported side-reactions for the Dmab,58 it has been widely used during last years'.59–61

Me

Dmab Et Fm Cam

Figure 5. The most significant C-terminal protecting groups removable under basic conditions.

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2.3. Removable Under Other Conditions

Other C-terminal protecting groups non-classified as acid- or basic-labile moieties have been employed because of their orthogonal feature which become them totally stable under mild acidic and basic conditions, being compatible with Fmoc/tBu and Boc/Bzl strategies. One of the preferred protector belonging to these non-classified group is the allyl, Al [removed under Pd(PPh3)4 (0.1 equiv) and PhSiH3 (10 equiv) as 62 scavenger in DCM, or Pd(PPh3)4 and morpholine as nucleophile in THF/DMSO/0.5 M HCl (2:2:1)63] (Fig. 6) used in SPPS64,65 and in solution. During years this carboxylic protector has aided to elongate peptides and this is the best proof of its fruitfulness in terms of orthogonality, thereby Al has been described as a compatible protector in those peptides which contain saccharides66,67 or lipids.68,69

Carboxylic acid protecting groups based on the benzyl function have introduced a broad range of versatility regarding the orthogonality. The most simple structure is the 70 benzyl, Bn (removed under cat. H2; or HF; or aq. NaOH; or TFMSA) (Fig. 6), mainly used in solution-phase, specifically in dipeptide synthesis.71–73 Nitrobenzyl derivatives were described as more stable towards acids as compared with the Bn moiety because of the deactivating effect of the nitro group. The most known principal protector based 45,74 on nitrobenzyl moiety is the para-nitrobenzyl, p-NB [removed under cat. H2; or tetrabutylammonium fluoride (TBAF) in THF, DMF or in DMSO; or SnCl2 in DMF; or

Na2S·H2O in H2O; or Na2S2O4 in aq. Na2CO3] (Fig. 6), described to reach linear peptide sequences75 and also to afford cyclic peptides.76,77 On the other hand, the methoxy•containing nitrobenzyl analogue 4,5-dimethoxy-2-nitrobenzyl, Dmnb78 (photolabile at 320 nm wavelength) (Fig. 6) which was designed as a totally orthogonal protector due to its already mentioned peculiar lability feature that becomes it stable under acidic conditions during SPPS.79 Finally, the 2-phenyl-2-trimethylsilyl, PTMSE80 81,82 (removed under TBAF·3H2O in DCM) (Fig. 6) described as a linker cleavable under almost neutral conditions by fluoride compounds, as well as the PTMSE has been reported as an effective carboxylic protecting group for certain peptide synthesis.83

PTMSE

Al

Bn P-NB Dmnb

Figure 6. The most significant C-terminal protecting groups removable under other conditions.

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(73) Maity, J.; Saha, P.; Gerling, U.; Lentz, D.; Koksch, B. An Approach for Simultaneous Synthesis of Cis- and Trans-3-Substituted Proline-Glutamic Acid Chimeras. Synthesis 2012, 44, 3063–3070. (74) Lammert, S. R.; Ellis, A. I.; Chauvette, R. R.; Kukolja, S. Azetidinone Antibiotics. 19. A Simple Method for the Removal of p-Nitrobenzyl Acid Protective Group. J. Org. Chem. 1978, 43, 1243–1244. (75) Namikoshi, M.; Kundu, B.; Rinehart, K. L. Use of Tetrabutylammonium Fluoride as a Facile Deprotecting Reagent for 4-Nitrobenzyl, 2,2,2-Trichloroethyl, and Phenacyl Esters of Amino Acids. J. Org. Chem. 1991, 56, 5464–5466. (76) Romanovskis, P.; Spatola, A. F. Preparation of Head-to-Tail Cyclic Peptides via Side- Chain Attachment: Implications for Library Synthesis. J. Pept. Res. 1998, 52, 356–374. (77) Royo, M.; Farrera-Sinfreu, J.; Solé, L.; Albericio, F. Four-Dimensional Orthogonal Solid- Phase Synthesis of New Scaffolds Based on Cyclic Tetra-Beta-Peptides. Tetrahedron Lett. 2002, 43, 2029–2032. (78) Lodder, M.; Golovine, S.; Laikhter, A. L.; Karginov, V. A.; Hecht, S. M. Misacylated Transfer RNAs Having a Chemically Removable Protecting Group. J. Org. Chem. 1998, 63, 794– 803. (79) Bourgault, S.; Létourneau, M.; Fournier, A. Development of Photolabile Caged Analogs of Endothelin-1. Peptides 2007, 28, 1074–1082. (80) Wagner, M.; Kunz, H. The 2-Phenyl-2-Trimethylsilyl)ethyl (PTMSE) Ester - A Carboxy Protecting Group Cleavable Under Neutral Conditions. Zeitschrift für Naturforsch. B - A J. Chem. Sci. 2002, 57, 928–936. (81) Wagner, M.; Kunz, H. The (2-Phenyl-2-Trimethylsilyl)ethyl-(PTMSEL) linker–A Novel Linker for the Solid-Phase Synthesis of Protected Peptides and Glycopeptides Cleavable with Fluoride. Angew. Chemie Int. Ed. 2002, 41, 317–321. (82) Lumbierres, M.; Palomo, J. M.; Kragol, G.; Waldmann, H. Solid-Phase Synthesis of Palmitoylated and Farnesylated Lipopeptides Employing the Fluoride-Labile PTMSEL Linker. Tetrahedron Lett. 2006, 47, 2671–2674. (83) Dziadek, S.; Espínola, C. G.; Kunz, H. Synthetic Glycopeptides for the Development of Antitumour Vaccines. Aust. J. Chem. 2003, 56, 519–543.

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Linear versus branched poly-lysine/arginine as polarity enhancer tags† Cite this: Org. Biomol. Chem., 2014, 12, 7194 Marta Paradís-Bas,a,b Maria Albert-Soriano,a Judit Tulla-Puche*a,b and Received 30th June 2014, a,b,c,d,e Accepted 30th July 2014 Fernando Albericio* DOI: 10.1039/c4ob01354a

www.rsc.org/obc

The design and synthesis of Lys- and Arg-containing peptides as more, the groups of Englebretsen,9 Wade,10 and Brimble11 fl – solubilizing tags were studied to evaluate their in uence on attached (Gly Arg)4 and (Lys)5 via a base linker to the C-termi- polarity. The relevance of spatial arrangement of polar groups, in nus of the poorly soluble peptide. Although Kuroda12 and co- α-orε-amino positions, was confirmed by chromatographic analy- workers reported that polyArg confers slightly more solubility sis of a rational PolyLys-based synthesized structure. The most than its Lys counterpart to the protein Bovine Pancreatic promising of the solubilizing tags here analyzed was conjugated to Trypsin Inhibitor-22 (BPTI-22), which contains 22 Ala residues, a commercial water-insoluble drug (indomethacin) as proof of we were intrigued whether this was also valid for a peptide and, concept. more importantly, by the contribution of the spatial arrange- ment disposition (linear vs. branched) to enhancing solubility. The synthesis, purification, and manipulation of biological To evaluate the polarity effect, several tags were assembled active molecules are often jeopardized by the poor polarity of stepwise by the solid-phase technique on a di-naphthylalanine the substrates, which is translated into low solubility for small [H–(Nal)2–NH2, 1] moiety, which was selected as a non-polar molecules, peptides, and proteins alike. In the case of small molecule. By means of an Fmoc/tBu strategy, all the peptide molecules, a clear example is the preparation of antibody drug sequences proposed (Fig. 1) were synthesized onto a Rink amide conjugates (ADCs). For the preparation of these molecules, the polystyrene resin using DIPCDI/OxymaPure® as coupling conjugation of the antibody and the drug in the final step reagents.13 All couplings were checked by the Kaiser test.14 Peptides must be performed in aqueous medium. With respect to pep- were analyzed by HPLC to determine the difference in polarity. tides, the solubility of these molecules can be increased by First, we performed a comparative study with the incorpor- introducing temporary chemical modifications such as N-(2- ation of four amino or four guanidinium groups, which were 1,2 acetoxy-4-methoxy)benzyl (acyl-Hmb) or O-acylisoacylpep- introduced into four peptides with distinct structures. The 3,4 tides; however, these strategies are not straightforward. amino groups were introduced through Lys, resulting in com- Another alternative is the introduction of solubilizing tags, pounds 2 (linear) and 8 (branched), while guanidinium groups mostly derived from oligoethyleneglycol or polycationic pep- were introduced through Arg, corresponding to peptides 3 and 5 tides. The former do not always render the desired solubility. 4 (both linear). Fig. 2a shows the superposed HPLC chromato- 6 7 8 The groups of Aimoto, Kent, and Brimble used (Arg)5/6 as a grams. As expected, all polycationic peptides conferred more part of the thioester moiety to increase the solubility of peptide fragments in a chemical ligation strategy. Further-

aInstitute for Research in Biomedicine Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain. E-mail: [email protected], [email protected]; Fax: +34 934037126; Tel: +34 934037088 bCIBER-BBN, Networking Centre for Address, Baldiri Reixac 10, 08028 Barcelona, Spain cDepartment of Organic Chemistry, Martí i Franquès 1, 08028 Barcelona, Spain dSchool of Chemistry & Physics, University of KwaZulu Natal, 4001 Durban, South Africa eSchool of Chemistry, Yachay Tech, Yachay City of Knowledge, 100119 Urcuquí, Ecuador †Electronic supplementary information (ESI) available: Materials and methods, characterization of synthesized peptides and solubility analysis. See DOI: 10.1039/c4ob01354a Fig. 1 Chemical structure of the peptides synthesized.

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Fig. 3 Chemical structure of a synthesized linear PolyLys peptide.

Fig. 2 Superposition of HPLC chromatographic profiles of: (a) first generation peptides (1, 2, 3, 4, 8); and (b) second generation peptides (5, 6, 7, 9). Study of polarity influence of amino and guanidinium groups in linear (2, 3, 4, 5, 6, 7) and branched (8, 9) Poly-AA sequences.

Fig. 4 Superposition of chromatogram analysis by HPLC of three lysine-based peptide sequences of linear (5)- and dendron (9, 10)-based – ff polarity to the (Nal)2 moiety. However, di erences were noted structures. Study of both α/ε-type amino groups influence and spatial depending on the nature of the tag. Thus, Lys-containing peptides arrangement disposition. induced greater enhancement of polarity than those holding Arg.

In addition, branched peptide 8 (tR =4.9min)showedaslightly 9 ε α 10 greater polarity than the linear peptide 2 (tR =5.1min). bears 4 - and 4 -, we synthesized the linear PolyLys In the second experiment, looking for a confirmation of the (Fig. 3), which contains 4ε- and 4α-amino groups similar to 9. first results, we increased the number of cationic groups to 8. The synthesis of 10, which sandwiched α- and ε-amide Three linear sequences matching peptides 5 (Lys), 6 and 7 bonds, was also performed using a Fmoc strategy by sequential (Arg) and one branched peptide 9 (Lys) were synthesized. addition of Fmoc-Lys(Boc)-OH and Boc-Lys(Fmoc)-OH. Analogous to the first set of peptides, PolyLys conferred Comparative HPLC analysis of 5, 9, and 10 (Fig. 4) revealed sig- higher polarity than PolyArg (Fig. 2b). Most importantly, the nificant polarity differences depending on the spatial arrange- 9 “ ” branched peptide ( , tR = 4.2 min) gave much more polarity ment disposition of the polar moieties. Thus, the two linear 5 than its equivalent linear peptide (5, tR = 6.8 min), thus sequences showed very similar retention times ( , tR = 6.8 min; confirming the trend observed with the first generation of 10, tR = 6.7 min). These times were higher than the most

peptides. “spherical” structure (9, tR = 4.2 min). Encouraged and intrigued by the large differences in the Once the chromatographic results confirmed that the behaviour of PolyLys compounds 5 and 9 and taking into “favorite” PolyLys structure (in terms of substantially increas- account that peptide 5 contains 7ε- and 1α-amino groups and ing the polarity) was the branch shaped moiety (9), a commercial

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Fig. 5 Indomethacin (11) and branched PolyLys peptide conjugated to indomethacin (12) compared by: (a) HPLC chromatography, elution at a

linear gradient of 20–90% MeCN containing 0.036% TFA in 0.045% aqueous TFA over 8 min; and (b) solubility in H2O (0.72 mM) of indomethacin (dispersion) and conjugated indomethacin (soluble).

drug was selected to be conjugated with branched PolyLys. de Catalunya (2009SGR 1024), and the Institute for Research in Indomethacin 11 (Fig. 5) is a non-polar drug which provided Biomedicine (Spain). We thank Dr Carmen Cuevas [Pharma- the water-insolubility feature suitable as a proof of concept to Mar SA, Madrid (Spain)] for fruitful discussions. test the polarity/solubility modulation of the PolyLys conju- gation. The ligation of both molecules was performed stepwise in the solid phase, by incorporating the drug on the side-chain Notes and references of the C-terminal Lys. Once the conjugated PolyLys–indo- 35 methacin was built up on the resin and after its release, 12 1 T. Johnson and M. Quibell, Tetrahedron Lett., 1994, , 463. (Fig. 5) was purified. HPLC analysis comparison between the 2 C. Hyde, T. Johnson, D. Owen, M. Quibell and 43 standard indomethacin and the conjugated PolyLys–indo- R. C. Sheppard, Int. J. Pept. Protein Res., 1994, , 431. methacin (Fig. 5a) revealed that the polarity of indomethacin 3 Y. Sohma, Y. Hayashi, M. Kimura, Y. Chiyomori, is impressively increased when it is combined with the A. Taniguchi, M. Sasaki, T. Kimura and Y. Kiso, J. Pept. Sci., 2005, 11, 441. branched PolyLys [retention time switches from (11) tR = 7.5 min to (12), t = 3.2 min]. 4 L. A. Carpino, E. Krause, C. D. Sferdean, M. Bienert and R 46 More importantly, the different solubility in water of the M. Beyermann, Tetrahedron Lett., 2005, , 1361. two molecules (Fig. 5b) gives the unequivocal evidence of the 5 A. I. Fernández-Llamazares, J. Adan, F. Mitjans, J. Spengler 25 enhancing polarity power of the branched PolyLys when a non- and F. Albericio, Bioconjugate Chem., 2014, , 11. 11 polar moiety is conjugated (see ESI† for details). 6 T. Sato, Y. Saito and S. Aimoto, J. Pept. Sci., 2005, , 410. In summary, linear PolyLys leads to a greater increase in 7 E. C. B. Johnson and S. B. H. Kent, Tetrahedron Lett., 2007, 48 polarity than its Arg counterpart. Moreover, this enhanced , 1795. 94 effect is much greater when the PolyLys adopts a branched 8 P. W. R. Harris and M. A. Brimble, Biopolymers, 2009, , arrangement. The conjugation of branched PolyLys with one 542. commercial drug (indomethacin) with intrinsic characteristics 9 D. R. Englebretsen and P. F. Alewood, Tetrahedron Lett., 37 of insolubility in water media is an example which confirms 1996, , 8431. the capacity of branched PolyLys to increase the polarity and 10 A. Belgi, M. A. Hossain, F. Shabanpoor, L. Chan, S. Zhang, most importantly the water solubility of a non-polar molecule. R. A. D. Bathgate, G. W. Tregear and J. D. Wade, Biochemis- 50 We envisage that a branched PolyLys—which in dendrimer try, 2011, , 8352. chemistry can be considered a dendron—will be crucial as a 11 S.-H. Yang, J. M. Wojnar, P. W. R. Harris, A. L. DeVries, polarity enhancer tag to facilitate the purification of peptides C. W. Evans and M. A. Brimble, Org. Biomol. Chem., 2013, 11 and/or the manipulation of peptides and small molecules. , 4935. 12 A. Kato, K. Maki, T. Ebina, K. Kuwajima, K. Soda and Y. Kuroda, Biopolymers, 2006, 85, 12. Acknowledgements 13 A. El-Faham, R. Subirós Funosas, R. Prohens and F. Albericio, Chem. – Eur. J., 2009, 15, 9404. The work was partially supported by MINECO (MARINMAB, 14 E. Kaiser, R. L. Colescot, C. D. Bossinge and P. I. Cook, IPT-2012-0198-09000), CICYT (CTQ2012-30930), the Generalitat Anal. Biochem., 1970, 34, 595.

7196 | Org. Biomol. Chem.,2014,12,7194–7196 This journal is © The Royal Society of Chemistry 2014 165

CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication III Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is © The Royal Society of Chemistry 2014

Electronic Supplementary Information

Linear versus Branched poly-Lysine/Arginine as Polarity Enhancer Tags

Marta Paradís-Bas,a,b Maria Albert-Soriano,a,b Judit Tulla-Puche,*a,b and

Fernando Albericio*a,b,c,d,e

a Institute for Research in Biomedicine Barcelona, Baldiri Reixac 10, 08028-Barcelona, Spain

b CIBER-BBN, Networking Centre for Address, Baldiri Reixac 10, 08028-Barcelona, Spain

c Department of Organic Chemistry, Martí i Franquès 1, 08028-Barcelona, Spain

d School of Chemistry & Physics, University of KwaZulu Natal, 4001-Durban, South Africa

e School of Chemistry, Yachay Tech, Yachay City of Knowledge, 100119.Urcuquí, Ecuador

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MATERIALS AND METHODS

Fmoc-L-Nal(1)-OH was purchased from Neosystem (Strasbourg, France) and the rest of the

Fmoc-L-AA-OH amino acids from Iris Biotech (Marktredwitz, Germany). Coupling reagents

most commonly used: DIPCDI, OxymaPure®, DIEA, and formic acid were from Sigma-Aldrich

(Schnelldorf, Germany), and other coupling reagents, such as PyBOP, HBTU and HOBt, for

difficult amino acid incorporations were from Iris Biotech. Solvents used in SPPS were peptide

synthesis-grade DMF and CH2Cl2 from Carlo Erba-SdS (Sabadell, Spain). For final acetylation

steps and cleavage Ac2O, TFA (HPLC-grade) and TIS were purchased from Sigma-Aldrich. The

solid support for all peptide syntheses was Fmoc-Rink amide AM resin (Polystyrene, loading

0.69 mmol/g) from Iris Biotech. Indomethacin drug was purchased at Tokyo Chemical Industry

Co. (TCI).

Analytical HPLC and HPLC/ESMS measurements used HPLC-grade MeCN purchased from

Carlo Erba-SdS (Sabadell, Spain). The analytical HPLC were performed on a Waters instrument

comprising a separation module (Waters 2695), an automatic injector (Waters 717 autosampler),

a photodiode array detector (Waters 2998), and a software system controller (Empower). The

reverse-phase C18 column (4.6  100 mm, 3.5 m, XBridge BEH130 from Waters, Cerdanyola

del Vallès, Spain) was used at flow rate of 1 mL/min and UV detection at 220 nm. Linear

gradients (specified in every peptide synthesis) over 8 min of eluent B (MeCN + 0.036% TFA)

into eluent A (H2O + 0.045% TFA) were used. Analytical HPLC/ESMS were performed on a

Waters Micromass ZQ spectrometer, comprising a separation module (Waters 2695), an

automatic injector (Waters 717 autosampler), a photodiode array detector (Waters 2998), and a

software system controller MassLynx v. 4.1). Linear gradients were performed over 8 min on a

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reverse-phase C18 column (3.9 × 150 mm, 5 μm) with MeCN (+ 0.07% formic acid) into

aqueous (+ 0.1% formic acid) at 0.3 mL/min, UV detection was at 220 nm and mass scans were

acquired in positive ion mode.

Purification of compound 12 was performed in a semi-prep Waters equipment with 600 Delta

system comprising a sample manager (Waters 2747) and a UV detector (Waters 2487) with a

dual absorbance selected at 254 and 220 nm was used. The software used was a MassLynx

version. A reverse-phase C18 column (XBridgeTM Prep BEH130, Waters: 19 × 100 mm, 5μm)

was used. Linear gradient of 15-90% MeCN containing 0.1% TFA into 0.1% TFA aqueous over

30 min. The tubes which contain the expected peptide were combined and lyophilized to afford

the expected pure product.

EXPERIMENTAL PROCEDURES

General peptide synthesis procedure. Peptides were synthesized manually in polypropylene

syringes fitted with a polyethylene porous disk. Solvents and soluble reagents were removed by

suction. The standard SPPS 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy was

followed using Fmoc-Rink amide AM resin (Polystyrene, loading 0.69 mmol/g) as a solid

support. All peptides, except the branched ones (8, 9, 10 and 12), were synthesized using the

following amino acids: Fmoc-L-Nal(1)-OH, Fmoc-L-Arg(Pbf)-OH, and Fmoc-L-Lys(Boc)-OH.

In the case of the branched peptides 8 and 9, Fmoc-L-Lys(Fmoc)-OH was used to build the

sequences while the common Boc-L-Lys(Boc)-OH · DCHA was used for the last coupling.

Compound 10 was achieved by means of an alternating coupling of Boc-L-Lys(Fmoc)-OH first

and Fmoc-L-Lys(Boc)-OH second until a total of seven Lys couplings was reached. The

branched Poly-Lys peptide conjugated to Indomethacin drug (12) was synthesized using the

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appropriated Lys orthogonal protecting groups to allow attachment of the drug to the peptide

chain. Compared with peptide 9, an extra Lys (Fmoc-L-Lys(Mtt)-OH) was introduced on the

resin as a first amino acid, where the drug would be attached through its side chain. Once this

amino acid was coupled, the Mtt group was removed by treating the resin with

TFA/triisopropilsilane (TIS)/CH2Cl2 (2:10:88) (2 × 5min, 1 × 20 min, 1 × 5 min). The

Indomethacin (11), through its carboxylic acid, was coupled following the standard coupling

system and the other Lys residues were coupled with the same protecting groups used to build

compound 9. The standard coupling system used in all peptide syntheses was Fmoc-AA-OH (3

equiv.) with N,N’-diisopropylcarbodiimide (DIPCDI)/ ethyl 2-cyano-2-(hydroxyimino)acetate

(OxymaPure®)13 (3 equiv., each) in DMF at room temperature for 1 h. The Kaiser14 test was used

to check the absence of free amino groups. When a positive test was obtained, the amino acid

was re-coupled using first Fmoc-AA-OH (3 equiv.) with N-[(1H-benzotriazol-1-yl)-

(dimethylamino)methylene] N-methylmethanaminium hexafluorophosphate (HBTU)/1-

hydroxybenzotriazole (HOBt) (3 equiv., each) and DIEA (6 equiv.) in DMF for 45 min; if the

coupling was still not completed a last re-coupling was performed with Fmoc-AA-OH (3 equiv.),

benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (3 equiv.) and

DIEA (6 equiv.) in DMF for 45 min. Resin washings after every coupling and after Fmoc

removal were performed using DMF (3 × 1 min), CH2Cl2 (3 × 1 min) and DMF (3 × 1 min).

Fmoc removal after every coupling was done by piperidine/DMF (1:4) treatment (1 × 1 min, 2 ×

5 min). When required (4 and 7), the final acetylation of compounds was carried out using

Ac2O/DIEA (10 equiv., each) in DMF for 30 min. After completion of peptide sequences, the

resin was washed with DMF (3 × 1 min) and CH2Cl2 (3 × 1 min), and the peptide was cleaved

from the resin by treatment with TFA/H2O (95:5) for 1 h, conditions which removes also the

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2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and tert-butyloxycarbonyl (Boc)

protecting groups. The cleavage time was increased to 4 h when the peptide contained Arg,

because of the Pbf protecting group. The mixture was filtered in the same type of syringe used

during peptide elongation, and the solvent was removed under N2 (g). The peptide was

precipitated with cold diethyl ether and centrifuged to remove the liquid phase. In the case of

compound 12 the peptide crude was subjected to a purification step and the expected product

was isolated with excellent purity (>99%). The crude peptides were dissolved in H2O/MeCN

(1:1), lyophilized, and characterized by HPLC and MS without further purification.

CHARACTERIZATION OF PEPTIDES

Peptide 1– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 5.73 min, 99.9% purity, gradient (%B): 25-40% over 8 min). MS (ESI): m/z

+ calcd. for C26H25N3O2, 411.19, found 412.17 [M + H] .

Peptide 2– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 5.14 min, 98.4% purity, gradient (%B): 20-35% over 8 min). MS (ESI): m/z

+ + calcd. for C44H61N9O5, 795.48, found 796.54 [M + H] , 398.95 [M + 2H] /2.

Peptide 3– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 5.61 min, 95.9% purity, gradient (%B): 20-35% over 8 min). MS (ESI): m/z

+ calcd. for C44H61N15O5, 879.49, found 880.54 [M + H] .

Peptide 4– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 5.64 min, 91.6% purity, gradient (%B): 20-35% over 8 min). MS (ESI): m/z

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+ + calcd. for C53H76N18O7, 1076.61, found 1078.71 [M + H] , 539.83 [M + 2H] /2, 360.29 [M +

3H]+/3.

Peptide 5– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 6.75 min, 98.2% purity, gradient (%B): 15-30% over 8 min). MS (ESI): m/z

+ + calcd. for C68H109N17O9, 1307.86, found 1309.00 [M + H] , 655.19 [M + 2H] /2.

Peptide 6– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 7.55 min, 86% purity, gradient (%B): 15-30% over 8 min). MS (ESI): m/z calcd.

+ + for C68H109N31O9, 1503.90, found 753.42 [M + 2H] /2, 502.00 [M + 3H] /3.

Peptide 7– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 7. 50 min, 96.6% purity, gradient (%B): 15-30% over 8 min). MS (ESI): m/z

+ + calcd. for C76H123N35O11, 1702.01, found 852.85 [M + 2H] /2, 568.67 [M + 3H] /3.

Peptide 8– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 4.91 min, 97.7% purity, gradient (%B): 20-35% over 8 min). MS (ESI): m/z

+ + calcd. for C44H61N9O5, 795.48, found 796.54 [M + H] , 398.88 [M + 2H] /2.

Peptide 9– A white solid was obtained after lyophilization. HPLC characterization showed one

main peak (tR = 4.20 min, 99.2% purity, gradient (%B): 15-30% over 8 min). MS (ESI): m/z

+ + calcd. for C68H109N17O9, 1307.79, found 1309.13 [M + H] , 655.26 [M + 2H] /2, 437.27 [M +

3H]+/3.

Peptide 10– A white solid was obtained after lyophilization. HPLC characterization showed

one main peak (tR = 6.66 min, 94.7% purity, gradient (%B): 15-30% over 8 min). MS (ESI): m/z

+ calcd. for C68H109N17O9, 1307.86, found 655.32 [M + 2H] /2.

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Peptide 12– A white solid was obtained after purification. HPLC characterization showed one

main peak (tR = 3.19 min, 99.9% purity, gradient (%B): 20-90% over 8 min). MS (MALDI-

+ TOF): m/z calcd. for C67H113ClN18O11, 1380.85, found 1381.96 [M + H] and 1403.95 [M +

Na]+.

SOLUBILITY ANALYSIS

The two common solvents used to peptide analysis [H2O and H2OMeCN (1:1)] were added,

separately, to both, Indomethacin and the branched PolyLys peptide conjugated to Indomethacin.

The different solubility of both samples in the mentioned solvents was observed (Fig. S1) and

also checked by HPLC analysis (Fig. S2 and S3). Two different chromatographic analyses were

performed: (a) addition of H2O to a known quantity of sample until reaching a fixed

concentration (0.72 mM), filtration of the solution (0.45 μm) and analysis by HPLC; (b) addition

of H2OMeCN (1:1) to a known quantity of sample until reaching a fixed concentration (0.72

mM), filtration of the solution (0.45 μm) and analysis by HPLC. Measurements of the

absorbance intensity obtained in HPLC in four cases (Fig. S2 and S3) allowed us to evaluate the

different solubility behavior of two samples in the analyzed solvents. In the case of Indomethacin

in H2O, the absorbance in HPLC was 0.14 (Fig. S2a); however in a more non-polar media

H2OMeCN (1:1), the solid was more soluble and, at the same concentration, showed a

substantial increase of absorbance until 0.54 (Fig. S2b). This experiment confirms the poor

solubility of Indomethacin in H2O. In case of branched PolyLys peptide conjugated to

Indomethacin, at the same concentration, the sample showed total solubility in H2O, confirmed

by the HPLC absorbance, which is in the same range in both solvents, in H2O and in

H2OMeCN (1:1) (Fig. S3).

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Fig. S1 Solubility trials in H2O (0.72 mM) of: (a) Indomethacin (right) compared with branched

PolyLys peptide conjugated to Indomethacin (left); (b) Indomethacin floating in aqueous media;

(c) some Indomethacin solid precipitated at the bottom of the glass flask.

Fig. S2 HPLC chromatograms of Indomethacin at 0.72 mM in: (a) H2O; (b) in H2OMeCN

(1:1). Elution at a linear gradient of 20-90% MeCN containing 0.036% TFA into 0.045%

aqueous TFA over 8 min.

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Fig. S3 HPLC chromatograms of branched PolyLys peptide conjugated to Indomethacin at 0.72

mM in: (a) H2O; (b) in H2O-MeCN (1:1). Elution at a linear gradient of 20-90% MeCN

containing 0.036% TFA into 0.045% aqueous TFA over 8 min.

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Letter

pubs.acs.org/OrgLett

Semipermanent C‑Terminal Carboxylic Acid Protecting Group: Application to Solubilizing Peptides and Fragment Condensation †,‡ ,†,‡ ,†,‡,§,∥,⊥ Marta Paradís-Bas, Judit Tulla-Puche,* and Fernando Albericio* † Institute for Research in Biomedicine (IRB Barcelona) Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain ‡ CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain § Department of Organic Chemistry, University of Barcelona, Martí i Franques̀ 1-11, 08028 Barcelona, Spain ∥ School of Chemistry & Physics, University of Kwazulu-Natal, Durban 4001, South Africa ⊥ School of Chemistry, Yachay Tech, Yachay City of Knowledge, Urcuquí 100119, Ecuador

*S Supporting Information

ABSTRACT: The 2-methoxy-4-methylsulfinylbenzyl alcohol (Mmsb-OH) safety-catch linker has been described as a useful tool to overcome two obstacles in peptide synthesis: the solubility and fragment condensation of peptides. The incorporation of the linker into an insoluble peptide target, thereby allowing the conjugation of a poly-Lys as a “solubilizing tag”, notably enhanced the solubility of the peptide. The selective conditions that remove that linker favored its incorporation as a semipermanent C-terminal protecting group, thereby allowing fragment condensation of peptides.

eptides with a large number of nonpolar amino acids, self- Scheme 1. Mmsb-OH Linker Structure P assembling tendency, and highly structured or merely difficult sequences remain a challenge in terms of synthesis. One of the proposed strategies used to attain the synthesis of such molecules tackles the common limitation of insufficient solubility. In recent years, several synthetic modes aimed to enhance solubility have been described. Most are focused on the disruption of β-sheet interactions, which is achieved by using mainly backbone amide protecting groups, such as − pseudoprolines1 or others based on substituted benzyl group2 4 Previous studies initiated by Englebretsen and Alewood9 “ ” or the depsipeptides grounded on the O−N intramolecular acyl proposed the (Gly-Arg)4 motif as a solubilizing tag linked to a 13 migration strategies,5 among others. Expensive presynthesized sequence by the glycolic acid, and later Englebretsen again, 14 fi building blocks or the limitation of purification conditions followed by Wade, described a ve Lys sequence to solubilize associated with these strategies highlight the need to explore peptides linked through the base-labile 4-hydroxymethyl- benzoic acid (HMBA) linker. Brimble15 also used that last new tools to facilitate such syntheses. Although less studied, “ ” another peptide approach to increase solubility consists of the linker connected to a six Arg solubilizing tag . However, the use of those linkers can lead to the formation of undesired incorporation of a temporary C-terminal cationic peptide aspartimide side products, which are favored by the basic fragment attached to the native sequence by a cleavable linker, “ ” cleavage conditions. To circumvent this issue and to increase known as a solubilizing tag . Although this kind of method has the solubility of a given peptide, here we attached the sequence fi 6−8 been used in the eld of recombinant proteins for years, to a six Lys mer, as a “solubilizing tag”, through the Mmsb-OH 9,10 only a few examples have been described for peptides. There linker (Scheme 2). The peculiarity of this linker is that, in is currently no robust synthetic tool to address the increasing addition to being compatible with Boc/Bzl and Fmoc/tBu solid demand for peptide targets with inherent insolubility. In order phase peptide synthesis (SPPS) strategies, as a safety-catch16,17 to open the spectrum of applications in this area, and taking linker its cleavage is mediated after a selective chemical advantage of our previous experience in enhancing peptide modification (in that case reductive acidolysis), thereby solubility,11 herein we sought to exploit the 2-methoxy-4- 12 methylsulfinylbenzyl alcohol (Mmsb-OH) linker (Scheme 1) Received: November 22, 2014 as a C-terminal semipermanent protecting group. Published: December 29, 2014

© 2014 American Chemical Society 294 DOI: 10.1021/ol5033943 Org. Lett. 2015, 17, 294−297 179 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication______IV

Organic Letters Letter

Scheme 2. Proposed Solubility Peptide Answer by Using a Scheme 3. Synthetic Design of Fragment Condensation to “Solubilizing Tag” Attached to the Mmsb-OH Linker Obtain the Fragment H-(22Phe-39Ser)-OH

Figure 2. HPLC chromatograms of peptide crude products with Figure 1. Structures and HPLC chromatograms, gradient from 10 to unprotected side chains after fragment condensation, gradient from 30 60% B, of standard Q11 2 (a) and solubilizing tag-linked Q11 peptide to 80% B (a); Fmoc removal, gradient from 10 to 60% B (b); and 1 (b). Solubility assays were done at 1 mg/mL in water. Mmsb removal, gradient from 10 to 60% B (c). avoiding basic conditions and consequently side reactions related to the same. Herein, we described for the first time its The Mmsb-OH linker was synthesized by optimizing the incorporation by the Fmoc/tBu SPPS strategy. The insoluble strategy proposed by Thennarasu and collaborators12 (see target sequence Q11 (Ac-Gln-Gln-Lys-Phe-Gln-Phe-Gln-Phe- details in the Supporting Information). Only one purification 18 Glu-Gln-Gln-NH2) was selected to explore our proposal. This step was required, and an overall yield of 28% was obtained. peptide is a self-assembling molecule with the capacity to form Initially, the solubilizing tag-linked Q11 peptide (1) was hydrogels. Extensive biomedical Q11 applications have been synthesized by SPPS (see details in the Supporting reported; therefore, synthetic methods to achieve the sequence Information) on a Rink-amide resin, after the coupling of six 19,20 are required. Fmoc-L-Lys(Boc)-OH amino acids using OxymaPure (3 equiv)

295 DOI: 10.1021/ol5033943 − 180 Org. Lett. 2015, 17, 294 297 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication______IV

Organic Letters Letter

most important applications: fragment condensation, where the Mmsb would act as a C-terminal protecting group. Large peptide sequences sometimes require the use of sophisticated methods to assemble the peptide fragments in solution in order to reach the final target. One of the most common strategies for this purpose is to prepare peptide fragments for ensuing coupling in solution.22 The need for an appropriate combination of protecting groups is crucial, and in this regard, the Mmsb-OH, as a carboxylic protecting group, is highly suitable for this strategy. We selected the well-known peptide Exenatide to test the conjugation. This compound is of relevance because of its use in diabetes therapy.23 Specifically, the synthesized sequence was the acid carboxylic version of C- terminal Exenatide (NH2-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly- Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH), which was as- sembled from two presynthesized peptide fragments (Scheme 3). The separation of the fragments, in terms of retrosynthetic analysis, was chosen between 31Pro and 32Ser. First, Pro is a cyclic amino acid that does not allow racemization during fragment condensation; second, we took advantage of preceding literature regarding Exenatide synthesis.24 Initially, peptide fragments Fmoc-(22−31)-OH (3) and H-(32−39)- Mmsb (4) were synthesized separately by the tBu/Fmoc SPPS strategy on CTC25 and Sieber26 resin (see Supporting Figure 3. MS (MALDI-TOF) spectra of Mmsb removal monitored for Information for details), respectively. Both resins allow peptide 6. Starting material (6) (red) and final product H-(22−39)- synthesis of peptides with fully protected side chains. After OH (blue). cleavage with a reduced amount of TFA (1% in the case of CTC and 3% in the case of Sieber), the two peptides were and DIPCDI (3 equiv) over 1 h. The Mmsb linker was lyophilized and analyzed by HPLC and MS, both techniques introduced under mild conditions with HOSu (1.5 equiv) and confirming the expected sequences were properly protected DIPCDI (1.5 equiv) to avoid multiple incorporations. To (see Figures S3 and S4 in the Supporting Information). complete the Q11 amino acid sequence, the same conditions Fragment condensation of the two peptides was performed used to couple the Lys were applied. After the final acetylation using H-(32−39)-Mmsb (1.5 equiv) and Fmoc-(22−31)-OH [by Ac O (5 equiv) and DIEA (5 equiv) over 30 min], the (1 equiv) coupled to PyBOP (1 equiv) and HOBt (1 equiv) at 2 − peptide was cleaved with TFA/TIS/H2O (38:1:1) for 1 h, pH 8 9 in DMF. The reaction was stopped at 7 h. After rendering the expected sequence with high purity (99.9%) lyophilizing the crude product (5), the expected peptide was (Figure 1b and Figure S2 in the Supporting Information), obtained with 92% HPLC purity (Figure 2a and Figure S5 in thereby confirming the stability of the Mmsb linker under the the Supporting Information). acidic cleavage conditions. Parallel to this synthesis, the Fmoc was removed with diethylamine to facilitate the carboxylic acid version of Q11, assigned as a standard (2), analysis, rendering the expected protected sequence (6) with was synthesized on the same type of resin using the same 91% HPLC purity (Figure 2b and Figure S6 in the Supporting coupling reagent conditions described before. After cleavage Information). Finally, in a one-pot reaction by reductive with TFA/TIS/H2O (38:1:1) for 1 h, the peptide was acidolysis, the side chains and the Mmsb C-terminal protecting precipitated with ether and lyophilized to afford the sequence, groups were removed (Figure 3) to afford the expected H-(22− also with high purity (99.4%) (Figure 1a and Figure S1 in the 39)-OH peptide fragment with 85% HPLC purity (Figure 2c Supporting Information). Although the HPLC chromatograms and Figure S9 in the Supporting Information). In this case, do not show big differences in polarity, the modified Q11 because of the presence of Trp (an amino acid that is known to sequence (Figure 1b) showed enhanced solubility, which was be susceptible to alkylation), diisopropyl sulfide was added as a attributed to the strategy used to attach the sequence to the scavenger.27 Again, the total stability of Mmsb during fragment “ ” Mmsb-(Lys)6 solubilizing tag . condensation and also its application in convergent strategies to In order to obtain the native Q11 from the solubilizing tag- attain large peptide sequences were demonstrated. linked Q11 peptide, the crude product was subjected to a In summary, we synthesized the Mmsb-OH linker in a fi fi reductive acidolysis treatment by NH4I (30 equiv) in neat TFA straightforward manner in ve steps and only one puri cation (1 mg/mL) for 30 min. The ensuing MS analysis confirmed the step. The linker was later incorporated into a peptide sequence total release of the Q11 sequence (Figure S8 in the Supporting by SPPS, the first time applied in the Fmoc/tBu strategy. Information) with high peptide purity (99.9%). Mmsb-OH has been exploited in two important aspects focused In addition to demonstrating the capacity of Mmsb to on the development of new tools for peptide synthesis. First, improve peptide properties, thereby facilitating its manipu- Mmsb-OH serves as a tool to enhance the solubility of lation, and motivated by the possibility to have in our hands a peptides, such as Q11, through the incorporation of a potential C-terminal protecting group, we wanted to contribute “solubilizing tag”. Second, based on the carboxylic acid with those already known carboxylic protecting groups for protection, the linker has allowed the synthesis of a large peptide synthesis.21 Thereby, we addressed one of the SPPS’s fragment of Exenatide by fragment condensation. This

296 DOI: 10.1021/ol5033943 − 181 Org. Lett. 2015, 17, 294 297 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication______IV

Organic Letters Letter

achievement opens the possibility of using the Mmsb linker (25) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J. Pept. strategy to synthesize other peptides with difficult sequences. Protein Res. 1991, 37, 513. (26) Sieber, P. Tetrahedron Lett. 1987, 28, 2107. ■ ASSOCIATED CONTENT (27) In the literature, the addition of Me2S as a scavenger in reductive 12,28,29 * acidolysis is described. We proved that diisopropyl sulfide, S Supporting Information which is less volatile, works also as a scavenger to prevent Trp Experimental procedures, NMR characterization of Mmsb alkylation. synthesis, HPLC and MS peptides characterization, and (28) Vilaseca, M.; Nicolas,́ E.; Capdevila, F.; Giralt, E. Tetrahedron solubility data. This material is available free of charge via the 1998, 54, 15273. Internet at http://pubs.acs.org. (29) Hackenberger, C. P. R. Org. Biomol. Chem. 2006, 4, 2291. ■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The work was partially supported by CICYT (CTQ2012- 30930), the Generalitat de Catalunya (2014SGR 137), and the Institute for Research in Biomedicine (IRB Barcelona). J.T.-P. is a Ramon y Cajal researcher (MINECO). ■ REFERENCES (1) Mutter, M.; Oppliger, H.; Zier, A. Makromol. Chem., Rapid Commun. 1992, 13, 151. (2) Johnson, T.; Quibell, M.; Owen, D.; Sheppard, R. C. J. Chem. Soc. Chem. Commun. 1993, 369. (3) Kiso, Y.; Tanaka, S.; Kimura, T.; Itoh, H.; Akaji, K. Chem. Pharm. Bull. 1991, 39, 3097. (4) Zahariev, S.; Guarnaccia, C.; Zanuttin, F.; Pintar, A.; Esposito, G.; Maravic,́ G.; Krust, B.; Hovanessian, A. G.; Pongor, S. J. Pept. Sci. 2005, 11, 17. (5) Sohma, Y.; Kiso, Y. ChemBioChem. 2006, 7, 1549. (6) Terpe, K. Appl. Microbiol. Biotechnol. 2003, 60, 523. (7) Wang, Z.; Li, H.; Guan, W.; Ling, H.; Wang, Z.; Mu, T.; Shuler, F. D.; Fang, X. Protein Expression Purif. 2010, 73, 203. (8) Kato, A.; Maki, K.; Ebina, T.; Kuwajima, K.; Soda, K.; Kuroda, Y. Biopolymers 2006, 85, 12. (9) Englebretsen, D.; Alewood, P. Tetrahedron Lett. 1996, 37, 8431. (10) Yang, S.-H.; Wojnar, J. M.; Harris, P. W. R.; DeVries, A. L.; Evans, C. W.; Brimble, M. A. Org. Biomol. Chem. 2013, 11, 4935. (11) Paradís-Bas, M.; Tulla-Puche, J.; Albericio, F. Chem.Eur. J. 2014, 20, 15031. (12) Thennarasu, S.; Liu, C.-F. Tetrahedron Lett. 2010, 51, 3218. (13) Choma, C. T.; Robillard, G. T.; Englebretsen, D. R. Tetrahedron Lett. 1998, 39, 2417. (14) Hossain, M. A.; Belgi, A.; Lin, F.; Zhang, S.; Shabanpoor, F.; Chan, L.; Belyea, C.; Truong, H.-T.; Blair, A. R.; Andrikopoulos, S.; Tregear, G. W.; Wade, J. D. Bioconjugate Chem. 2009, 20, 1390. (15) Harris, P.; Brimble, M. Synthesis 2009, 20, 3460. (16) Kenner, G.; McDermott, J.; Sheppard, R. Chem. Commun. 1971, 426, 636. (17) Patek,́ M.; Lebl, M. Tetrahedron Lett. 1991, 32, 3891. (18) Collier, J. H.; Messersmith, P. B. Adv. Mater. 2004, 16, 907. (19) Jung, J. P.; Jones, J. L.; Cronier, S. A.; Collier, J. H. Biomaterials 2008, 29, 2143. (20) Hosseinkhani, H.; Hong, P.-D.; Yu, D.-S. Chem. Rev. 2013, 113, 4837. (21) Alsina, J.; Albericio, F. Biopolymers 2003, 71, 454. (22) Mergler, M. Methods Mol. Biol. 1994, 35, 287. (23) McCormack, P. L. Drugs 2014, 74, 325. (24) Brichard, M.-H.; Cauvin, J.-M.; Devijver, C.; Droz, A.-S.; Gilles, P.; Giraud, M.; Latassa, D.; Rekai, E. D.; Varray, S.; Albericio, F.; Paradís-Bas, M. WO 2011006644 A2, 2011.

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Supporting Information

Semipermanent C-Terminal Carboxylic Acid Protecting Group: Application to Solubilizing Peptides and Fragment Condensation

†,‡ ,†,‡ ,†,‡,§, ⊥ Marta Paradís-Bas, Judit Tulla-Puche,* and Fernando Albericio* ǁ,

†Institute for Research in Biomedicine (IRB Barcelona) Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain; ‡CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain; §Department of Organic Chemistry, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain; ǁSchool of Chemistry & Physics, University of Kwazulu-Natal, Durban 4001, South Africa; ⊥School of Chemistry, Yachay Tech, Yachay City of Knowledge, Urcuquí 100119, Ecuador

Table of Contents

Abbreviations...... S2

Materials and General Methods...... S2

Experimental section

Synthesis of Mmsb-OH Linker...... S3

Solid-Phase Peptide Synthesis...... S6

A) Synthesis of Q11 and the "solubilizing tag" Mmsb-analogue...... S6

B) Synthesis of Exenatide Fragments Fmoc-(22-31)-OH and H-(32-39)-Mmsb.... S8

Fragment Condensation to Afford Exenatide Fragment H-(22-39)-OH...... S10

Peptide Analysis...... S11

Peptide Analysis after Mmsb Removal...... S15

Solubility Analysis of Q11 and its Mmsb-analogue...... S16

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Abbreviations

AA, amino acid; tBu, tert-butyl; CTC, 2-chlorotrityl chloride; DIPCDI, N,N’- diisopropylcarbodiimide; DIEA, N,N’-diisopropylethylamine; DMF, N,N’- dimethylformamide; DMAP, N,N’-dimethylpyridin-4-amine; Fmoc, 9-fluorenylmethyl carbamate; HFIP (1,1,1,3,3,3-hexafluoro-2-propanol); HOBt, 1-hydroxybenzotriazole; HOSu, N-hydroxysuccinimide; OxymaPure, ethyl 2-cyano-2-(hydroxyimino)acetate; PyBOP, benzotriazol-1-yloxytri(pyrrolidino)phosphonium hexafluorophosphate; TIS, triisopropylsilane ; TFA, trifluoroacetic acid.

Materials and General Methods

All Fmoc-L-AA-OH, HOBt and the CTC resin were purchased from Iris Biotech. Sieber resin and DMAP were purchased from Novabiochem. Coupling systems such as DIPCDI, OxymaPure, PyBOP, HOSu and DIEA were from Sigma-Aldrich. Solvents

used in SPPS, CH2Cl2 and DMF were peptide synthesis-grade from Carlo Erba-SdS. Acetylation (acetic anhydride), cleavage reagents [TIS and TFA (HPLC-grade)] and also the solvent HFIP were purchased from Sigma-Aldrich. Solid-Phase syntheses were performed manually in polypropylene syringes fitted with a polyethylene porous disc. Solvents and soluble reagents were removed by suction.

Peptide crudes were dissolved commonly in H2O/MeCN (1:1) to be analyzed by HPLC, MALDI-TOF MS or HPLC-ESI MS. When peptides were not soluble in those conditions, the solvent used to dissolve them is specified in the peptide analysis section. HPLC characterizations were carried out on Waters instrument comprising a separation module (Waters 2695) and a photodiode array detector (Waters 2998) with a software system controller Empower. The analytical column used to characterize the synthesized Mmsb-OH and the peptide crudes was a reverse-phase C18 column (XBridgeTM BEH130, 4.6 x 100 mm, 3.5 µm). Common column temperature analysis used was 25 ºC and when T = 60 ºC was required, it has been specified in the peptide analysis section. UV detection was measured at 220 nm for peptides and measured by a maximum of absorbance average in case of Mmsb-OH synthesis. Flow rate was at 1 mL/min. The eluent system used in all HPLC conditions was based in linear gradients

of eluent B (MeCN + 0.036% TFA) into eluent A (H2O + 0.045% TFA) over 8 min. Mass analyses of peptides were performed by the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) on an Applied Biosystems Voyager-DE RP instrument, using α-cyano-4-hydroxycinamic acid as a

matrix dissolved in H2O/MeCN (1:1) containing 0.1% of TFA. Mass analyses of synthesized Mmsb and its intermediates were carried out on a Waters instrument in an

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analytical RP-HPLC-ESMS with a Micromass ZQ spectrometer comprising a separation module (Waters 2695), an automatic injector (Waters 717 autosampler), a photodiode array detector (Waters 2998) and a software system controller MassLynx v. 4.1. The analytical column was a reverse-phase C18 column (SunFireTM, 2.1 x 100 mm, 5 µm). UV detection was measured at 220 nm, mass scans were acquired in positive ion mode. Flow rate was at 0.3 mL/min. The eluent system used was based on

linear gradients of eluent MeCN (+ 0.07% formic acid) into H2O (+ 0.1% formic acid) over 8 min. 1H and 13C NMR spectra were recorded on a Varian MERCURY 400 (400 MHz for 1H NMR, 100 MHz for 13C NMR) spectrometer. Chemical shifts (δ) are expressed in parts per million downfield from tetramethylsilyl chloride. Coupling constants are expressed in Hertz.

Experimental Section

Synthesis of Mmsb-OH Linker

Ethyl 2-[(3-methoxyphenyl)thio]acetate

3-methoxythiophenol (1.77 mL, 14.3 mmol) was dissolved in CH2Cl2 (7 mL) and after cooling the solution in an ice-water bath, ethyl 2- bromoacetate (1.77 mL, 15.7 mmol) was added. Then, triethylamine (2.19 mL, 14.3 mmol) was added dropwise (to avoid dimerization of starting material) and the mixture was warmed to room temperature and stirred for 30 min (monitored by HPLC). After completion of the reaction, the mixture was washed

with H2O (3 × 10 mL) and brine (3 × 10 mL). The organic layer was dried (MgSO4), filtered and then, the solvent was removed under reduced pressure to afford the expected compound as a yellow oil (3.07 g, 95%), pure enough (99%) to be used in the following reaction without further purification.

HPLC analysis (tR = 3.61 min; gradient from 50% to 100% B over 8 min). 1 H NMR (400 MHz, CDCl3; Me4Si): δ 7.21 (t, J = 7.9 Hz, 1H), 6.99 – 6.94 (m, 2H), 6.76

(ddd, J = 8.3, 2.4, 1.0 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H, CH2), 3.79 (s, 3H, OCH3), 3.64

(s, 2H, SCH2), 1.24 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 169.78 (C), 160.00 (C), 136.50 (C), 129.96 (CH),

121.86 (CH), 115.03 (CH), 112.87 (CH), 61.72 (CH2), 55.42 (OCH3), 36.65 (CH2),

14.23 (CH3).

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Ethyl 2-[(4-formyl-3-methoxyphenyl)thio]acetate

Freshly Vilsmeier reagent (6 equiv) was prepared as follows: in a 100 mL round-bottomed flask, phosphorous oxychloride (8 mL, 89.7

mmol) was added followed by dry CH2Cl2 (19 mL) under an argon atmosphere. The solution was cooled to 10 ºC with an ice-water bath

and a solution of dry DMF (5.11 mL, 65.8 mmol) in dry CH2Cl2 (19 mL) was added dropwise. After the Vilsmeier reagent was prepared, a solution of ethyl 2-

[(3-methoxyphenyl)thio]acetate (3.38 g, 14.9 mmol) in dry CH2Cl2 (4.7 mL) was added slowly. The ice bath was removed and the solution was heated under reflux to 60 ºC and stirred for 24 h. After the reaction mixture was cooled to room temperature, the mixture was poured carefully into crushed ice and the product was extracted with

CH2Cl2 (3 × 30 mL). The organic layers were combined, dried (MgSO4), filtered and then, the solvent was evaporated under reduced pressure. Residual DMF was

removed by co-evaporation with toluene, CH2Cl2 and diethyl ether consecutively. The resulting pale brown solid contains as a major isomer the expected product in 66.7% purity. A portion of this crude (2.5 g) was subjected to purification by CombiFlash Rf 200 system (reverse phase C18 silica, linear gradient from (B/A) 5:95 to 45:55 over 45 min, then isocratic 45:55 over 15 min) to yield the expected compound, after lyophilization, as a white solid (974 mg, non optimized 39% yield).

HPLC analysis (tR = 2.89 min; gradient from 50% to 100% B over 8 min). The expected

product shows an HPLC-MS (ESI): m/z calcd for C12H14O4S: 254.06, found: 183.21 [M + + H] . The main impurity detected (HPLC tR = 2.71 min; gradient from 50% to 100% MeCN over 8 min) corresponds to the other isomer [ethyl 2-[(2-formyl-5-

methoxyphenyl)thio]acetate], HPLC-MS (ESI): m/z calcd for C12H14O4S: 254.06, found: 254.85 [M + H]+. 1 H NMR (400 MHz CDCl3; Me4Si): δ 10.34 (s, 1H, CHO), 7.73 (d, J = 8.2 Hz, 1H), 6.95

(d, J = 1.6 Hz, 1H), 6.92 (dd, J = 8.2, 1.6 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H, CH2), 3.92

(s, 3H, OCH3), 3.74 (s, 2H, SCH2), 1.26 (t, J = 7.1 Hz, 3H, CH3). 13 C NMR (100 MHz, CDCl3): δ 188.86 (CHO), 169.16 (C), 161.87 (C), 146.15 (C),

129.08 (CH), 122.70 (C), 118.76 (CH), 109.85 (CH), 62.13 (CH2), 55.88 (OCH3), 34.84

(CH2), 14.24 (CH3).

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Ethyl 2-[(4-(hydroxymethyl)-3-methoxyphenyl)thio]acetate

Fresh sodium triacetoxyborohydride reducing agent (3 equiv) was

prepared as follows: NaBH4 (223 mg, 5.9 mmol) was suspended in ethyl acetate (45 mL), stirred and cooled with an ice-water bath. Then, glacial acetic acid (1 mL, 6 mmol) was added slowly and stirred for 10 min. After the reducing agent was prepared, the previously prepared aldehyde (500 mg, 1.9 mmol) was dissolved in the minimum quantity of ethyl acetate and the solution was added dropwise (over 20 min). The mixture was warmed to room temperature and stirred for 2 h (monitored by HPLC). After completion of the reaction,

the mixture was washed with H2O (3 × 50 mL) and brine (3 × 50 mL). The organic layer

was dried (MgSO4), filtered and then, the solvent was removed under reduced pressure to afford the expected compound as a white solid (475.7 mg, 95%), pure enough (98%) to be used in the following reaction without further purification.

HPLC analysis (tR = 4.07 min; gradient from 30% to 80% B over 8 min). 1 H NMR (400 MHz, CDCl3; Me4Si): δ 7.21 (d, J = 8.0 Hz, 1H), 6.99 – 6.95 (m, 2H), 4.63

(s, 2H, CH2OH), 4.17 (q, J = 7.1 Hz, 2H, CH2), 3.85 (s, 3H, OCH3), 3.63 (s, 2H, SCH2),

1.24 (t, J = 7.1 Hz, 3H, CH3). 13 C NMR (100 MHz, CDCl3): δ 169.84 (CO), 157.65 (C), 135.81 (C), 129.23 (CH),

128.21 (C), 121.95 (CH), 112.06 (CH), 61.76 (CH2), 61.67 (CH2), 55.56 (OCH3), 36.91

(SCH2), 14.25 (CH3).

Ethyl 2-[(4-(hydroxymethyl)-3-methoxyphenyl)sulfinyl]acetate

Acetic acid (27 mL) was added to dissolve the previously prepared alcohol (464.5 mg, 1.8 mmol). A solution of aqueous hydrogen peroxide (30%) (5.89 mL, 196.5 mmol) was slowly added to the reaction mixture while stirring until total conversion1 (confirmed by HPLC). The mixture was poured onto crushed ice and the product

was extracted with CH2Cl2 (3 × 40 mL). The organic layers were combined and dried

(MgSO4). The organic solvent was removed under reduced pressure. Acetic acid was removed by co-evaporation with toluene, dichloromethane and diethyl ether to afford the expected compound as a white solid (490 mg, 99.3%). This product was pure enough (99%) to be used in the following reaction without further purification.

1 Golchoubian, H.; Hosseinpoor, F. Molecules 2007, 12, 304.

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HPLC analysis (tR = 2.98 min; gradient from 20% to 50% B for 8 min). 1 H NMR (400 MHz, CDCl3; Me4Si): δ 7.46 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 1.5 Hz, 1H),

7.14 (dd, J = 7.7, 1.5 Hz, 1H), 4.71 (s, 2H, CH2OH), 4.17 (q, J = 7.1 Hz, 2H, CH2), 3.92

(s, 3H, OCH3), 3.73 (dd, J = 76.4, 13.7 Hz, 2H, SOCH2), 1.24 (t, J = 7.1 Hz, 3H, CH3). 13 C NMR (100 MHz, CDCl3): δ 164.87 (CO), 158.22 (C), 143.53 (C), 133.47 (C), 128.82

(CH), 116.59 (CH), 105.22 (CH), 62.25 (CH2), 62.11 (CH2), 61.16 (CH2), 55.92 (OCH3),

14.18 (CH3).

2-[(4-(hydroxymethyl)-3-methoxyphenyl)sulfinyl]acetic acid [Mmsb-OH]

The compound previously prepared (464.8 mg, 1.7 mmol) was

dissolved in THF/H2O (2:1) (20 mL) and LiOH (215 mg, 5.1 mmol) was slowly added, and stirred for 30 min (monitored by HPLC). The mixture was acidified with the aid of HCl (0.2 M) to pH 1 and the

product was extracted with CH2Cl2 (3 × 25 mL). Organic layers were

combined, dried (MgSO4), filtered, and the solvent was removed under reduced pressure affording the expected compound as a white solid (79.4%). This product was pure enough (99%) to be used on solid-phase peptide synthesis without further purification. The overall yield to convert 3-methoxythiophenol to Mmsb-OH was 28%.

HPLC analysis (tR = 3.90 min; gradient from 5% to 40% B for 8 min). The main product

corresponds to the expected final Mmsb-OH, HPLC-MS (ESI): m/z calcd for C10H12O5S: 244.04, found: 244.96 [M + H] +. 1 H NMR (400 MHz, CD3OD; Me4Si): δ 7.60 (d, J = 7.8 Hz, 1H), 7.37 – 7.27 (m, 2H),

4.89 (s, 2H, CH2OH), 4.67 (s, 2H, SCH2), 3.92 (s, 3H, OCH3). 13 C NMR (101 MHz, CD3OD): δ 168.05 (COOH), 158.91 (C), 143.38 (C), 135.48 (C),

129.18 (CH), 117.52 (CH), 106.47 (CH), 62.33 (CH2), 59.87 (CH2), 56.22 (OCH3).

Solid-Phase Peptide Synthesis

A) Synthesis of Q11 and the "solubilizing tag" Mmsb-analogue

The two peptides: the standard Q11 (2) and the solubilizing tag-linked Q11 (1) were synthesized by using standard Fmoc/tBu strategy on aminomethyl-ChemMatrix resin

(50 mg, 0.66 mmol/g). The resin was conditioned by washings with TFA/CH2Cl2 (1:99)

(5 x 0.5 min), CH2Cl2 (5 x 0.5 min), DIEA/CH2Cl2 (1:19) (5 x 0.5 min) and CH2Cl2 (5 x 0.5 min). Previously to amino acids incorporation, the resin was functionalized by coupling the commercial [3-(4-hydroxymethylphenoxy)-propionic acid linker, in case of standard Q11 (2); and Fmoc-Rink amide linker, in case of solubilizing tag-linked Q11

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(1)]. Both the commercial linker and the common amino acids (Fmoc-L-AA-OH) were incorporated using 3 equiv, DIPCDI (3 equiv) and OxymaPure (3 equiv) in DMF, with a 5-min preactivation and a total coupling time of 1 h. After every incorporation the resin

was washed with DMF (3 × 1 min) and CH2Cl2 (3 × 1 min). Then, a Kaiser test was carried out to check which amino acid was not completely coupled. In the case of incomplete couplings, the same conditions were applied to repeat the incorporation. Fmoc removal was performed with piperidine/DMF (1:4) (25 mL/g resin, 1 × 1 min, 2 ×

5 min), followed by resin washings with DMF (3 × 1 min), CH2Cl2 (3 × 1 min) and DMF (3 × 1 min). The cycle of AA coupling/Fmoc removal was performed until complete elongation of peptides.

Incorporation of Mmsb-OH linker

The incorporation of the synthesized Mmsb-OH linker for the solubilizing tag-linked Q11 was carried out under mild conditions: Mmsb (1.5 equiv), N-hydroxysuccinimide (HOSu) (1.5 equiv) and DIPCDI (1.5 equiv) in DMF for 30 min. Kaiser test was carried out to check its complete incorporation.

Ester bond formation on previously incorporated Mmsb-OH linker

Ester bond formation between the amino acid and the alcohol functionalized linkers [commercial 3-(4-hydroxymethylphenoxy)-propionic acid or synthesized Mmsb-OH] was carried out using the symmetrical anhydride conditions: Fmoc-AA-OH (4 equiv),

DIPCDI (2 equiv) catalyzed by DMAP (0.2 equiv) in CH2Cl2. After 3 h of reaction, a second incorporation using fresh reagents, was performed for 15 h.

Final acetylation and cleavage/global deprotection of peptides

After Fmoc removal of the last amino acid, acetylation of N-terminal amino acid was carried out with acetic anhydride (10 equiv) and DIEA (10 equiv) for 30 min, followed by

resin washings with DMF (3 × 1 min), CH2Cl2 (3 × 1 min). Kaiser test confirmed the completed reaction. Then, the resin was subjected to global deprotection/cleavage by

treatment with TFA/TIS/H2O (38:1:1) for 1 h. The mixture was partially evaporated under reduced pressure and the peptides were precipitated with cold diethyl ether. The liquid layer was removed by centrifugation and the solids were washed with cold diethyl

ether to give white solids that were dissolved in H2O/MeCN (1:1) and lyophilized. The two peptide crudes were analyzed by HPLC and MS (MALDI-TOF) (see Figure S1 and S2).

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Removal of Mmsb moiety

The Mmsb moiety of solubilizing tag-linked Q11 (1) was removed under the following

conditions: 1 mg of peptide crude 1 was dissolved in neat TFA (1 mL/mg) and NH4I (1.7 mg, 11.9 µmol) was added and stirred at room temperature. The reaction was monitored by MALDI-TOF MS (see Figure S7) and, after 60 min, the reaction was stopped, although by MALDI-TOF no starting material was observed after 30 min. The work-up was accomplished by filtering the solution to remove iodine impurities, evaporating the solvent under reduced pressure, further precipitation and washings of the peptide with cold diethyl ether. Characterization of the final unprotected peptide was performed by HPLC and MS (MALDI-TOF) (see Figure S8).

B) Synthesis of Exenatide fragments Fmoc-(22-31)-OH and H-(32-39)-Mmsb

1) Synthesis of Fmoc-(22-31)-OH (3)

N-terminal fragment Fmoc-(22-31)-OH (3) was synthesized by using standard Fmoc/tBu strategy on 2-chlorotrityl chloride resin (CTC) (100 mg, 1.55 mmol/g). The

resin was conditioned by washings with DMF (3 × 2 min) and CH2Cl2 (3 × 1 min). The first Fmoc-Pro-OH (1 mmol/g resin) was incorporated in presence of DIEA (10 equiv), which was added in two portions: first 1/3 of the volume was added and after 10 min, the remaining 2/3. The mixture was allowed to react for 45 min. Next, a capping step with MeOH (0.4 mL/g resin) was performed for 10 min. Fmoc removal was performed with piperidine/DMF (1:4) (25 mL/g resin, 1 × 1 min, 2 × 5 min), followed by resin

washings with DMF (3 × 1 min), CH2Cl2 (3 × 1 min) and DMF (3 × 1 min). Other Fmoc- L-AA-OH were coupled using 3 equiv, DIPCDI (3 equiv) and OxymaPure (3 equiv) in DMF, with a 5-min preactivation and a total coupling time of 1 h. After each

incorporation, the resin was washed with DMF (3 × 1 min) and CH2Cl2 (3 × 1 min). Then, a Kaiser test was carried out to check which amino acid was not completely coupled. In the case of incomplete couplings, the same conditions were applied to repeat the incorporation. The cycle of AA coupling/Fmoc removal was performed until the elongation of peptide was complete. The last Fmoc group was maintained at the N-terminal position for fragment condensation step.

Cleavage of N-terminal peptide fragment

The peptide 3 was cleaved from the resin with TFA/CH2Cl2 (1:99) (8 × 0.5 min) and

CH2Cl2 (3 × 1 min), and collected onto H2O to avoid side-chain deprotection. The solvent was removed under reduced pressure and finally, the peptide was lyophilized,

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affording the fully protected peptide 3 as a white solid, which was analyzed by HPLC and MS (MALDI-TOF) (see Figure S3).

2) Synthesis of H-(32-39)-Mmsb (4)

The C-terminal fragment H-(32-39)-Mmsb (4) was synthesized by using standard Fmoc/tBu strategy on Fmoc-Sieber-amide aminomethyl polystyrene resin (100 mg, 0.69 mmol/g). The resin was conditioned by washings with DMF (3 × 1 min) and

CH2Cl2 (3 × 1 min). Fmoc removal of Sieber linker was performed with piperidine/DMF (1:4) (25 mL/g resin, 1 × 1 min, 2 × 5 min), followed by resin washings with DMF (3 × 1

min), CH2Cl2 (3 × 1 min) and DMF (3 × 1 min). Common amino acids (Fmoc-L-AA-OH) were incorporated using 3 equiv, DIPCDI (3 equiv) and OxymaPure (3 equiv) in DMF, with a 5-min preactivation and a total coupling time of 1 h. After every incorporation, the

resin was washed with DMF (3 × 1 min) and CH2Cl2 (3 × 1 min). Then, a Kaiser test was carried out to check which amino acid was not completely coupled. In the case of incomplete couplings, the same conditions were applied to repeat the incorporation.

Incorporation of Mmsb-OH linker

After removing the Fmoc group of Sieber resin, the incorporation of synthesized Mmsb linker was performed with the same conditions described for the Q11 Mmsb-containing linker analogue: Mmsb-OH (1.5 equiv), N-hydroxysuccinimide (HOSu) (1.5 equiv) and DIPCDI (1.5 equiv) in DMF for 30 min. Kaiser test was carried out to check its complete incorporation.

Ester bond formation on previously incorporated Mmsb-OH linker

Ester bond formation between the amino acid and the alcohol group of Mmsb-OH linker was carried out using the same conditions described by Q11 Mmsb-analogue by using symmetrical anhydride conditions: Fmoc-AA-OH (4 equiv), DIPCDI (2 equiv), catalyzed

by DMAP (0.2 equiv) in CH2Cl2. After 3 h of reaction, a second incorporation for 15 h was performed.

Cleavage of the C-terminal peptide fragment

The peptide was cleaved from the resin with TFA/CH2Cl2 (1:32) (8 × 1 min) and CH2Cl2

(3 × 1 min), and collected onto H2O to avoid side-chain deprotection. The solvent was removed under reduced pressure and finally, the peptide was lyophilized affording the fully protected peptide 4 as a white solid, which was analyzed by HPLC and MS (MALDI-TOF) (see Figure S4).

191 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication IV

Fragment Condensation to Afford Exenatide Fragment H-(22-39)-OH

Synthesis of Fmoc-(22-39)-Mmsb (5)

To a solution of the N-terminal fragment Fmoc-(22-31)-OH (3) (11.5 mg, 6.1 µmol) with HOBt (1 mg, 6.1 µmol) in DMF (150 µL), a solution of C-terminal fragment H-(32-39)- Mmsb (4) (10 mg, 9.2 µmol) in DMF (40 µL) was added. Then, the coupling reagent PyBOP (4.1 mg, 7.9 µmol) dissolved in DMF (40 µL) was added to the mixture and finally the pH was adjusted with DIEA to 8-9. The mixture was stirred at room temperature for 7 h (monitored by HPLC). After completion of the reaction, the mixture was directly lyophilized to afford the Exenatide fragment Fmoc-(22-39)-Mmsb (5) as a white solid, which was analyzed by HPLC and MS (MALDI-TOF) (see Figure S5).

Synthesis of H-(22-39)-Mmsb (6)

The Fmoc removal of Fmoc-(22-39)-Mmsb (5) (7 mg, 2.45 µmol) was performed in

solution by using diethylamine (5 µL, 73.5 µmol) in CH2Cl2 (30 µL). The mixture was stirred at room temperature for 4 h. After the solvent removal, the peptide was washed

with diethyl ether (3 x 1 mL) to dissolve and remove the dibenzofulvene adduct. H2O was added to wash the solid (3 x 1 mL) and to allow the removal of the residual HOBt from the previous reaction. The white solid was lyophilized and analyzed by HPLC and MS (MALDI-TOF) (see Figure S6).

Removal of the Mmsb moiety: Synthesis of Exenatide Fragment H-(22-39)-OH

1 mg of peptide 6 was dissolved in neat TFA (1 mL/mg). In this case diisopropyl sulfide (10 µL, 1%, 68.8 µmol) was added because of the presence of tryptophan in the

sequence (susceptible to suffer alkylation) and then, NH4I (1.6 mg, 11.4 µmol) was added and stirred at room temperature. The reaction was monitored by MALDI-TOF MS and after 45 min the reaction was stopped, although no starting material was observed by MALDI-TOF after 15 min. The work-up was accomplished by filtering the solution to remove iodine impurities, evaporating the solvent under reduced pressure, further precipitation and washings of the peptide with cold diethyl ether. Characterization of the final unprotected peptide was performed by HPLC and MS (MALDI-TOF) (see Figure S9).

192 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication IV

Peptide Analysis

Standard Q11 (2)

HPLC analysis of Standard Q11 crude 2 after global deprotection/cleavage from the

resin, and further dissolution in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (tR = 5.39 min; gradient from 10% to 60% B over 8 min) shows one main product (99.4%, HPLC purity) which corresponds to the expected Standard Q11 (2), MS (MALDI-TOF) (Figure S1): + + m/z calcd for C70H98N18O21: 1526.71, found: 1527.77 [M + H] , 1549.76 [M + Na] .

Mass (m/z) Figure S1. MALDI-TOF MS spectrum of Standard Q11.

Solubilizing tag-linked Q11 (1)

HPLC analysis of Solubilizing tag-linked Q11 crude 1 after global deprotection/cleavage from the resin, and further dissolution in 1,1,1,3,3,3-hexafluoro-

2-propanol (HFIP) (tR = 4.91 min; gradient from 10% to 60% B over 8 min) shows one main product (99.9%, HPLC purity) which corresponds to the expected Solubilizing tag-

linked Q11 (1), MS (MALDI-TOF) (Figure S2): m/z calcd for C116H181N31O30S 2520.33, found: 2521.60 [M + H]+. Other m/z found: 828.68 [M + H]+ is associated to a decomposition of Mmsb moiety linked to the peptide which releases the sulfonyl

derivative and the Ac-(Lys)6-NH2 (calcd for C38H77N13O7 827.61) caused by MALDI-TOF analysis (detected also in other Mmsb-analogues).

Mass (m/z)

Figure S2. MALDI-TOF MS spectrum of Solubilizing tag-linked Q11.

193 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication IV

N-terminal fragment Fmoc-(22-31)-OH (3) HPLC analysis (Figure S3a) of fragment Fmoc-(22-31)-OH crude (3), with fully

protected side-chains, dissolved in HFIP (tR = 3.51 min; gradient from 90% to 100% B over 8 min) shows one main product (97%, HPLC purity). However, it was required to

treat some portion with TFA/TIS/H2O (38:1:1) for 1 h to remove the protecting groups and facilitate the detection by MS. HPLC analysis (Figure S3b) of fragment Fmoc-(22-

31)-OH crude with unprotected side-chains (tR = 5.91 min; column temperature at 60 ºC; gradient from 30% to 80% B over 8 min) shows one main product (99%, HPLC purity) which corresponds to the Fmoc-(22-31)-OH, MS (MALDI-TOF) (Figure S3c): + + m/z calcd for C71H91N13O16 1381.67, found: 1382.81 [M + H] , 1404.82 [M + Na] . Some impurity, not observed in the HPLC, which corresponds to the peptide without the Fmoc protecting group is detected by MS (MALDI-TOF) (Figure S3c): m/z calcd for + + C56H81N13O14 1159.60, found: 1160.7 [M + H] , 1182.71 [M + Na] .

a )

tR (min)

b ) c )

Mass (m/z) tR (min) Figure S3. Analysis of N-terminal fragment Fmoc-(22-31)-OH: HPLC chromatogram with protected side- chains (a); HPLC chromatogram with unprotected side-chains (b); and MALDI-TOF MS spectrum of unprotected side-chains fragment (c).

C-terminal fragment H-(32-39)-Mmsb (4) HPLC analysis (Figure S4a) of fragment H-(32-39)-Mmsb crude 4 with fully protected

side-chains (tR = 6.16 min; column temperature at 60 ºC; gradient from 10% to 60% B over 8 min) shows one main product (99%, HPLC purity) which corresponds to the expected C-terminal fragment H-(32-39)-Mmsb, MS (MALDI-TOF) (Figure S4b): m/z + + calcd for C51H81N9O15S 1091.56, found: 1092.57 [M + H] , 1114.59 [M + Na] and 1130.57 [M + K]+. Other m/z found: 1036.51 is associated to a decomposition of Mmsb moiety linked to the peptide which releases the sulfonyl derivative (56 units less, calcd

for C49H80N8O14S 1036.55) caused by MALDI-TOF analysis (detected also in other Mmsb-analogues).

194 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication IV

a ) b )

Mass (m/z) tR (min) Figure S4. Analysis of C-terminal fragment H-(32-39)-Mmsb with fully protected side-chains: HPLC chromatogram (a); and MALDI-TOF MS spectrum (b).

Fragment Condensation: Fmoc-(22-39)-Mmsb (5)

HPLC analysis (Figure S5a) of fragment Fmoc-(22-39)-Mmsb crude 5, with fully

protected side-chains, dissolved in HFIP (tR = 3.68 min; column temperature at 60 ºC; gradient from 80% to 100% B over 8 min) shows one main product (84%, HPLC purity).

However, it was required to treat some portion with TFA/TIS/H2O (38:1:1) for 1 h to remove the protecting groups and facilitate the detection by MS. HPLC analysis of

fragment Fmoc-(22-39)-Mmsb crude with unprotected side-chains (tR = 5.23 min; column temperature at 60 ºC; gradient from 30% to 80% B over 8 min) shows one main product (92%, HPLC purity) which corresponds to the Fmoc-(22-39)-Mmsb, MS

(MALDI-TOF) (Figure S5b): m/z calcd for C110H146N22O30S 2287.03, found: 2288.32 [M + H]+. Other m/z found: 2232.54 is associated to a decomposition of the Mmsb moiety linked to the peptide which releases the sulfonyl derivative (56 units less, calcd for

C108H145N21O29S 2232.02) caused by MALDI-TOF analysis (detected in other Mmsb-

analogues). The main impurity isolated at (5%, HPLC purity tR = 5.89 min; column temperature at 60 ºC; gradient from 30% to 80% B over 8 min) corresponds to the N-terminal fragment [Fmoc-(22-31)-OH], MS (MALDI-TOF) (Figure S5c): m/z calcd for + + C71H91N13O16 1381.67, found: 1382.83 [M + H] , 1404.85 [M + Na] and 1420.83 [M + K]+. a )

tR (min)

b ) c)

Figure S5. Analysis of Fmoc-(22-39)-Mmsb: HPLC chromatogram with protected side-chains (a); MALDI- TOF MS spectrum of collected tR = 5.23 min product with unprotected side-chains (b); and MALDI-TOF MS spectrum of collected tR = 5.89 min product with unprotected side-chains (c).

195 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication IV

Fragment Condensation: H-(22-39)-Mmsb (6)

HPLC analysis (Figure S6a) of fragment H-(22-39)-Mmsb crude 6, with fully protected

side-chains, dissolved in HFIP (tR = 5.22 min; column temperature at 60 ºC; gradient from 50% to 100% B over 8 min) shows one main product (91%, HPLC purity).

However, it was required to treat some portion with TFA/TIS/H2O (38:1:1) for 1 h to remove the protecting groups and facilitate the detection by MS. HPLC analysis of

fragment H-(22-39)-Mmsb crude with unprotected side-chains (tR = 4.77 min; column temperature at 60 ºC; gradient from 10% to 60% B over 8 min) shows one main product (85.5%, HPLC purity) which corresponds to the H-(22-39)-Mmsb, MS (MALDI- + TOF) (Figure S6b): m/z calcd for C95H136N22O28S 2064.96, found: 2066.22 [M + H] . Other m/z found: 2010.40, and its corresponding 2032.41 [M + Na]+, are associated to a decomposition of the Mmsb moiety linked to the peptide which releases the sulfonyl

derivative (minus 56 units, calcd for C93H135N21O27S 2009.95) caused by MALDI-TOF

analysis (detected in other Mmsb-analogues). The main impurity (3.7%, HPLC purity tR = 5.56 min; column temperature at 60 ºC; gradient from 10% to 60% B over 8 min) corresponds to the N-terminal fragment [H-(22-31)-OH], MS (RP-HPLC-ESI): m/z calcd + for C56H81N13O14 1159.60, found: 1161.25 [M + H] .

a ) b )

Mass (m/z) tR (min)

Figure S6. Analysis of H-(22-39)-Mmsb: HPLC chromatogram with protected side-chains (a); and MALDI- TOF MS spectrum of product with unprotected side-chains (b).

196 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication IV

Analysis of Peptides after Mmsb Removal

Solubilizing tag-linked Q11

t = 0 min

t = 30 min

t = 60 min

Mass (m/z)

Figure S7. Monitoring the Mmsb removal of solubilizing tag-linked Q11 by MALDI-TOF MS. Starting material containing the Mmsb linker (blue circle); Intermediate of the reaction with the reduced sulfoxide from Mmsb (orange triangle); and Expected Q11 peptide without Mmsb (green rectangle).

HPLC analysis (Figure S8) of solubilizing tag-linked Q11, after Mmsb removal

treatment, dissolved in HFIP (tR = 4.37 min; gradient from 10% to 60% B over 8 min) shows one main product (99%, HPLC purity) which corresponds to the expected Q11,

MS (MALDI-TOF) (Figure S8): m/z calcd for C70H98N18O21 1526.71, found: 1527.91 [M + + + H] and 1549.91 [M + Na] .

Figure S8. HPLC chromatogram and MALDI-TOF MS spectrum of Q11 released from solubilizing tag- linked Q11 after Mmsb removal.

197 CHAPTER______3: Peptide-Based Solubilizing Tag Strategies ______Publication IV

Fragment Condensation: H-(22-39)-OH

HPLC analysis (Figure S9) of H-(22-39)-OH, after Mmsb removal treatment, dissolved

in HFIP (tR = 4.49 min; column temperature at 60 ºC; gradient from 10% to 60% B over 8 min) shows one main product (85%, HPLC purity) which corresponds to the expected

H-(22-39)-OH, MS (MALDI-TOF) (Figure S9): m/z calcd for C85H125N21O25 1839.91, + + found: 1841.11 [M + H] , 1863.09 [M + Na] . The impurity (3.4%, HPLC purity tR = 5.40 min; column temperature at 60 ºC; gradient from 10% to 60% B over 8 min) corresponds to the N-terminal fragment [H-(22-31)-OH], MS (RP-HPLC-ESI): ): m/z + calcd for C56H81N13O14 1159.60, found: 1160.68 [M + H] .

Mass (m/z)

Figure S9. MALDI-TOF MS spectrum of Exenatide fragment H-(22-39)-OH after Mmsb removal.

Solubility Analysis of Q11 and its Mmsb-analogue

Standard Q11 and the Solubilizing tag-linked Q11

1 mg of Standard Q11 crude (2) and 1 mg of Solubilizing tag-linked Q11 crude (1) were

dissolved separately in H2O (1 mL) and compared. It was clearly observed that Standard Q11 is not soluble and, on the contrary, the Solubilizing tag-linked Q11 is completely soluble in water.

198

GENERAL CONCLUSIONS

General Conclusions

"Difficult peptides", as well as those sequences that aggregate in solution, are widely present in a large range of areas such as biochemistry, nanomaterials or even medicine. The structural behavior is its most distinguished feature but unfortunately, is also the responsible to produce synthetic drawbacks. In response to the constantly increasing demand of finding straightforward methodologies to reach these peptides, we have contributed to overcome these difficulties. The strategies proposed and evaluated in the present thesis have expanded the scope of tools addressed to solubilize peptides not only during SPPS, but also after their elongations. The main conclusions, organized by chapters are the following:

Chapter 1. The RADA-16 self-assembling peptide, described as one of the most valuable sequences for its biomedicine applications, has been extensively studied in the present thesis. Three solid-phase peptide strategies were selected to address the RADA-16 synthesis. First, the most commonly used, the stepwise methodology; and later the two convergent approaches: the solid-phase and the fragment condensation in solution. The four times repeating Arg-Ala-Asp-Ala sequence comprised in this peptide was crucial for the retro-synthetic design of both convergent strategies. Fragment condensation in solution of two fully protected segments resulted in the most fruitful strategy to synthesize the RADA-16 peptide. Development of a new work- up protocol was essential to achieve the final peptide with a satisfactory purity. Several HPLC conditions of commercial RADA-16 samples were studied allowing to find the most significant parameters to achieve an accurate characterization, thus contributing to the current methods for the analysis of other similar sequences.

Chapter 2. The design of a new backbone amide protecting group, the Mmsb, was developed as a "safety-catch" protector in order to disrupt the β-sheet interactions present in "difficult peptides". The solid-phase incorporation of the Mmsb into a sequence was preceded by the synthesis of Mmsb α-amino protected alanine [Fmoc- N(Mmsb)-Ala-OH]. The safety-catch property of this sulfoxide derivative protecting group was confirmed by evaluating its resistance to acidic cleavage conditions, and its subsequent acid-labile conversion after reduction, to the thioether. Initially, Decaalanine was the sequence of choice to verify the synthetic benefits attributed to the new backbone amide protection, and the structural modifications induced by Mmsb were confirmed by circular dichroism. The RADA-16 synthesis served as a second "difficult peptide" example to demonstrate, not only improvements on the synthesis, but also the enhancement of the solubility in solution which allowed its HPLC purification. Finally, the synthesis of the challenging self-assembling Aβ(1-42) peptide

201 General Conclusions evidenced that the Mmsb strategy permits to overcome synthetic and also purification troubles, even for large "difficult peptides". Chapter 3. Several short cationic peptides based on Lys or Arg residues were synthesized and conjugated to a non-polar di-naphthylalanine moiety to evaluate their effect in terms of polarity. Linear and branched poly-Lys/Arg designed as solubilizing tags provided the following polarity features: (i) linear poly-Lys resulted more polar than its homologue poly-Arg; (ii) branched poly-Lys contributed to increase the polarity in a greater range than the linear form; (iii) amino-type (α or ε) exposed to the media influenced on a minor extent than the spatial distribution of those amino groups. The permanent ligation of a known insoluble drug to a branched poly-Lys solubilizing tag provided a proof of concept to demonstrate the tag contribution to enhance the solubility and polarity of the drug. The Mmsb structure used in the second chapter was applied as a linker (Mmsb-OH). The first proposed application of this handle consisted of a temporary connection between the non-soluble Q11 sequence and a linear poly-Lys solubilizing tag. Enhancement of solubility, as well as the conjugated solubilizing tag resistance towards acidolytic cleavage, permitted the accurate peptide characterization. Reductive acidolysis released the peptide target in the similar manner than the Mmsb backbone amide protector. The second application of Mmsb-OH led to the obtaining of a new C-terminal protecting group. Stability of Mmsb to cleavage conditions allowed reaching a fully protected peptide fragment, to be further conjugated in solution to another protected sequence. Fragment condensation of these sequences was carried out successfully and, the subsequent protecting groups removal rendered the expected peptide, validating the Mmsb-OH as a new C-terminal protector.

202

RESUM

CAPÍTOL 1: Avaluació Sintètica per a la Obtenció d'un "Pèptid Difícil" Resum

Capítol 1: Avaluació Sintètica per a la Obtenció d'un "Pèptid Difícil"

La propietat d'autoensamblar que presenten certes molècules ha estat valorada en el camp de la medicina i la tecnologia per a la cerca de nous nano-materials.1 La característica d'organització molecular que es dóna en aquests productes s'ha avaluat amb elevat interès, sent els més rellevants aquells destinats a la biomedicina.2 Algunes de les aplicacions clíniques que s'han dut a terme amb aquests materials han estat per exemple en la distribució de fàrmacs dins l'organisme,3 en la regeneració de teixits,4 per a l'ajut de cristal·lització de proteïnes5 o per facilitar la internalització en cèl·lules.6 La medicina imposa a aquests productes a ser forçosament biocompatibles, així que la comunitat científica s'ha fixat en els pèptids com a clars candidats, en tant que compleixen aquest requisit. A la natura es troben exemples de pèptids que poden autoensamblar, i aquests han servit com a model per al disseny de noves seqüències amb la finalitat que presentin aquesta característica.7 A part de ser biocompatibles, els pèptids permeten una senzilla combinació i modificació sintètica de la seqüència d'AAs (AAs) de manera que, de novo, es poden obtenir cadenes peptídiques que presentin autoensamblatge en solució.

A nivell molecular, s'ha pogut observar que un dels principals mecanismes a través del qual certs pèptids presenten la tendència a autoensamblar, es basa en la formació d'interaccions de tipus làmina-β. Aquestes associacions es donen quan el pèptid es troba totalment desprotegit i en solució, succeint tant a nivell intra- o com inter- molecular. De manera que, ja sigui entre diferents cadenes de pèptid, com entre una mateixa cadena, aquestes interaccions provoquen inicialment una associació en làmines-β, que en alguns casos es pot arribar a estendre en l'espai tridimensional donant supra-estructures com les fibres. La característica que presenten els pèptids que autoensamblen, considerada com un avantatge per crear nous materials, resulta ser un inconvenient pel què fa a la seva manipulació en solució.

Per altra banda, quan les forces d'unió làmina-β d'una seqüència es produeixen durant la síntesi del pèptid, ja sigui en fase sòlida o en solució, aquesta se l'anomena "pèptid difícil".8,9 Aquestes seqüències concretament presenten enllaços de pont d'hidrogen entre l'esquelet de la cadena peptídica, específicament entre el protó de l'amida i l'oxigen del carbonil. Les dificultats durant el procés conegut de síntesi de pèptids en fase sòlida (SPPS) succeeixen perquè el suport polimèric sobre el qual creix la seqüència no evita la formació d'interaccions entre els enllaços amida. Per definició no tots els pèptids difícils d'obtenir són "pèptids difícils", però sempre es donarà aquesta relació a la inversa. Tot i que a la literatura s'han descrit estructures peptídiques que

205 CAPÍTOL 1: Avaluació Sintètica per a la Obtenció d'un "Pèptid Difícil" Resum

s'engloben dins d'aquest conjunt, no és fàcil a priori tenir la certesa que una determinada seqüència serà un pèptid d'aquest tipus. S'han identificat, en base a les dificultats sintètiques, una sèrie de conceptes comuns a tots els "pèptids difícils":10,11 la constant impossibilitat d'incorporar certs AAs, malgrat l'augment del nombre d'intents; l'acusat problema sintètic quan la funcionalització de la resina és elevada; llargues o incomplertes desproteccions del grup amino abans de cada acoblament; i complicacions considerables quan hi ha AAs que contenen cadenes laterals voluminoses.

Entre les seqüències classificades a la literatura com a pèptids que autoensamblen en destaca un subgrup que s'anomenen pèptids amfifílics, ja que tenen una part de l'estructura que es comporta com a hidrofòbica i una altra part com a hidrofílica.5,12 Alhora, els pèptids amfifílics se subdivideixen en un altre grup menor que es coneix com pèptids d'autoensamblatge iònics. La majoria de membres d'aquest tipus són seqüències no naturals sintetitzades de novo, de manera que es composen per AAs amb cadenes laterals carregades positiva i negativament en una mateixa seqüència. La complementarietat de càrregues afavoreix substancialment l'autoensemblatge, motiu pel qual aquests pèptids han estat extensament estudiats. Concretament, pertanyent en aquest subgrup es troba el pèptid conegut com RADA-16, dissenyat junt amb altres seqüències similars pel professor Zhang l'any 200013 i que ha estat subjecte d'estudi en aquest capítol. En concret, aquest pèptid també presenta interaccions de tipus làmina-β durant la seva síntesi, així que es pot dir que és una seqüència que s'agrupa dins dels coneguts com "pèptids difícils".

El RADA-16 [Ac-(RADA)4-NH2] (Fig. 1), com el seu nom abreujat indica, és una seqüència de 16 AAs amb una repetició de quatre vegades el tetrapèptid Arg-Ala-Asp- Ala. Precisament, aquesta continua alternança de càrregues (positiva atribuïda a l'Arg i negativa atribuïda a l'Asp) és el què confereix al pèptid la seva elevada tendència a formar l'autoensamblatge. Recents estudis detallats sobre la seva estructura terciària o quaternària han ajudat a entendre que les cadenes del RADA-16 es disposen en forma de làmina-β paral·leles que s'ensamblen exposant dues cares definides. Aquestes dues parts defineixen dos plans, en tant que pèptid amfifílic, un és l'hidrofòbic (format per les Ala) i l'altre l'hidrofílic (format per l'alternança dels dos AAs carregats, l'Arg i l'Asp). Quan diferents cadenes del RADA exposen aquests plans en un mateix medi, inevitablement s'orienten en l'espai tridimensional de manera que els dos plans hidrofòbics s'encaren entre sí i els plans carregats se situen contraposant les càrregues positives amb les negatives. Degut a la organització d'aquest pèptid en solució, s'ha descrit la seva formulació com a hidrogel, concretament en un 99.5% de contingut

206 CAPÍTOL 1: Avaluació Sintètica per a la Obtenció d'un "Pèptid Difícil" Resum

d'aigua i l'addició d'una solució salina que afavoreix el seu autoensamblatge. Aquesta composició es comercialitza des del 2005,14 inicialment per l'empresa BD Bioscience que l'ha anomenat PuraMatrix,® i posteriorment per altres empreses que la ofereixen amb diferents graus de puresa i amb altres noms comercials. Aquest compost, tot i no ser directament un fàrmac, s'ha utilitzat en estudis mèdics per tal de facilitar l'alliberament de fàrmacs en cèl·lules, com a regeneració de nombrosos teixits, o fins i tot en tractaments combinats pel càncer en cèl·lules i en assajos in vivo.15

Fruit de totes les aplicacions, que es troben en augment dins la biomedicina associades al RADA-16, així com la seva consideració com a bon exemple de "pèptid difícil", en aquest capítol s'ha abordat la seva síntesi en fase sòlida, la seva purificació i caracterització. Com a resposta a la manca en la literatura d'un mètode sintètic eficient amb una detallada caracterització per al RADA-16, s'ha fet el següent estudi, també amb l'objectiu d'aportar noves eines sintètiques extensibles a altres "pèptids difícils" de característiques semblants.

Figura 1. Estructura del RADA-16.

Tot i que la SPPS de manera seqüencial és la primera estratègia utilitzada per a la obtenció de cadenes peptídiques llargues, s’han anat desenvolupat variants d’aquest mètode on es combina la SPPS i el tradicional mètode de síntesi en solució. Dins de l’objectiu principal d’obtenció del pèptid RADA-16 amb elevada puresa s’han establert les següents estratègies convergents: la condensació de fragments en fase sòlida16,17 i la condensació de fragments en solució.18 La primera es fonamenta en la obtenció del pèptid sencer en fase sòlida mitjançant la incorporació d’un segment peptídic d'aquest prèviament sintetitzat en fase sòlida. Per altra banda, la segona estratègia és portada a terme mitjançant la unió en solució de dos fragments peptídics sintetitzats prèviament de manera independent en fase sòlida.

Inicialment, mitjançant la via seqüencial, tant la manual com l'automàtica, es va obtenir la síntesi del RADA-16 amb un valor de puresa que s'utilitzarà com a referència per avaluar l'eficiència les altres estratègies proposades. Tot i la necessitat d’incorporar més d’una vegada certs AAs, el pèptid RADA-16 es va obtenir amb una puresa per

207 CAPÍTOL 1: Avaluació Sintètica per a la Obtenció d'un "Pèptid Difícil" Resum

HPLC del 65-75%, amb la massa esperada identificada per espectrometria de masses MALDI-TOF. L’anàlisi de les impureses detectades per HPLC del cru peptídic va mostrar que aquestes tenien unes característiques de polaritat molt semblants al pèptid objectiu, trobant-se molt properes al producte desitjat pel què fa a temps d’elució en el cromatograma, fet que suposa una gran barrera a l’hora de purificar.

A través de la via de condensació de fragments en fase sòlida, aprofitant la naturalesa de la seqüència del RADA-16, format de 4 fragments peptídics exactament iguals units consecutivament fins a 4 vegades, es va escollir el fragment peptídic següent: Fmoc-Arg(Pbf)-Ala-Asp(tBu)-Ala-OH (F1, Fig. 2). Aquest pèptid es va sintetitzar en fase sòlida i un cop escindit de la resina mantenia els seus grups protectors a les cadenes laterals d'aquells AAs funcionalitzats; la seva puresa va ser del 99% per HPLC. Tenint en compte que F1 presenta cert impediment estèric es va decidir evitar la seva incorporació directa a la resina realitzant l'acoblament del primer tetrapèptid seqüencialment, fent que el segon tetrapèptid (F1) es pogués incorporar sense problemes. Les dificultats d’introducció d’aquest es van veure incrementades a mesura que anava creixent el pèptid, de manera que el RADA-16 només es va obtenir amb un 46% de puresa.

Condensació de Fragments en Fase Sòlida

Cl Resina CTC NH2 Resina Rink ChemMatrix

Elongació del pèptid Elongació del pèptid Fmoc-Arg(Pbf)-Ala-Asp(tBu)-Ala-O Eliminació final del grup Fmoc

H-Arg(Pbf)-Ala-Asp(tBu)-Ala-NH (Peptidil-resina I) 1% TFA (desancorament)

i) Incorporació del F1 Fmoc-Arg(Pbf)-Ala-Asp(tBu)-Ala-OH ii) Eliminació del grup Fmoc (Fragment F1)

H-[Arg(Pbf)-Ala-Asp(tBu)-Ala]2-NH (Peptidil-resina II)

i) Incorporació del F1 ii) Eliminació del grup Fmoc

H-[Arg(Pbf)-Ala-Asp(tBu)-Ala]3-NH (Peptidil-resina III) i) Incorporació del F1 ii) Eliminació del grup Fmoc

H-[Arg(Pbf)-Ala-Asp(tBu)-Ala]4-NH (Peptidil-resina IV) i) Acetilació ii) 95% TFA

Ac-(RADA)4-NH2

Figura 2. Esquema sintètic de l'estratègia convergent de condensació de fragments en fase sòlida.

208 CAPÍTOL 1: Avaluació Sintètica per a la Obtenció d'un "Pèptid Difícil" Resum

Per últim, la via de condensació de fragments en solució va requerir d’una manera raonable i acurada una divisió d'aquest pèptid en els punts més òptims, que serien la unió de dos únics fragments de 8 AAs cadascun (Fig. 3). Els dos fragments peptídics,

Ac-[Arg(Pbf)-Ala-Asp(tBu)-Ala]2-OH (F2, Fig. 3) i H-[Arg(Pbf)-Ala-Asp(tBu)-Ala]2-NH2 (F3, Fig. 3), es van sintetitzar en gran escala (0.8 mmol) en fase sòlida. Les condicions per a la condensació en solució van ser equimolars i amb el sistema d’agents d’acoblament hexafluorofosfat de benzotriazol-1-il-N-oxi-tris(pirrolidino)fosfoni (PyBOP)/ N,N'-diisopropiletilamina (DIEA) amb l'additiu 1-hidroxi-7-azabenzotriazol (HOAt). Aquesta estratègia, junt amb les condicions descrites, va ser la via més òptima per a obtenir el pèptid desitjat amb una puresa considerablement superior a l’obtinguda amb les altres estratègies. Per altra banda, les impureses presents al cromatograma d’HPLC no es trobaven properes en temps al pèptid RADA-16, fet significant a l’hora de purificar un pèptid amb tendència a l’auto-ensamblatge. En aquest cas, la dificultat es va trobar en l’eliminació d’impureses, com els agents d’acoblament, sobretot l’additiu HOAt, que es va solucionar realitzant rentats del cru peptídic amb tert-butilmetiléter, obtenint-ne una puresa final del RADA-16 per HPLC del 85%.

Condensació de Fragments en Solució

Cl Resina CTC NH2 Resina Sieber

Elongació del pèptid Elongació del pèptid Acetilació final Eliminació final del grup Fmoc

Ac-[Arg(Pbf)-Ala-Asp(tBu)-Ala]2-O H-[Arg(Pbf)-Ala-Asp(tBu)-Ala]2-NH

1% TFA (desancorament) 3 % TFA (desancorament)

Ac-[Arg(Pbf)-Ala-Asp(tBu)-Ala]2-OH + H-[Arg(Pbf)-Ala-Asp(tBu)-Ala]2-NH2 (Fragment F2) (Fragment F3)

Ac-[Arg(Pbf)-Ala-Asp(tBu)-Ala]4-NH2

95% TFA (desprotecció global)

Ac-(RADA)4-NH2

Figura 3. Esquema sintètic de l'estratègia convergent de condensació de fragments en fase sòlida.

Per tal de purificar el RADA-16, es va pensar en un disseny acurat basat en extraccions que pogués aprofitar la diferència de solubilitat dels diferents productes segons la polaritat del dissolvent. Comparat amb el mètode estàndard utilitzat per a la purificació de pèptids per HPLC semi-preparatiu, aquest nou procediment proposat va permetre obtenir el RADA-16 amb una puresa del 90%, notablement millorada respecte la inicial.

209 CAPÍTOL 2: Un nou Grup Protector per a Amides de Cadena Peptídica Resum

En paral·lel a l'avaluació d'estratègies sintètiques per obtenir el RADA-16 es va realitzar un treball d'optimització de paràmetres per caracteritzar aquest pèptid per HPLC, ja que el seu autoensamblatge en dificulta el tractament en solució. Sis mostres d'aquest pèptid proporcionades per cases comercials diferents van ser utilitzades per a dur a terme el següent estudi. Els factors avaluats van ser: la dissolució de la mostra, la concentració injectada a l'equip, el gradient cromatogràfic, la temperatura de la columna, així com la fase mòbil. Aquest estudi va demostrar la importància d'aquests paràmetres quan es vol caracteritzar un pèptid que presenta autoensamblatge, de manera que es va obrir el ventall de factors a tenir en compte per analitzar pèptids amb aquesta característica.

En aquest capítol es conclou que s'han avaluat diferents estratègies sintètiques en fase sòlida per a la obtenció del pèptid RADA-16, combinant tant les més comunament utilitzades com altres que combinen la condensació de fragments en fase sòlida o en solució. S'ha establert que la manera d'aconseguir aquest "pèptid difícil" amb la màxima puresa és mitjançant la condensació de dos fragments del RADA-16 en solució. L'eliminació de certes impureses del cru peptídic s'ha pogut realitzar amb un posterior protocol d'extraccions i rentats en diferents dissolvents que han permès purificar el pèptid sense la necessitat d'utilitzar l'HPLC semi-preparatiu. Estudis de cromatografia líquida amb mostres comercials del RADA-16 han permès identificar aquells paràmetres més crucials a tenir en compte quan s'han d'analitzar pèptids que autoensamblen en solució.

Capítol 2: Un nou Grup Protector per a Amides de Cadena Peptídica

Els anteriorment descrits "pèptids difícils" suposen un repte sintètic perquè la seva tendència a formar estructures tipus làmina-β és una barrera que impedeix que els grups funcionals que han de formar l'enllaç amida ho puguin fer de manera assequible. Aquest inconvenient es dóna tant en la síntesi de pèptids en solució com en la SPPS, ja que, com s'ha esmentat, aquestes interaccions organitzades es donen entre els grups amida de l'esquelet peptídic, presents en les dues estratègies. En els dos tipus de processos aquesta problemàtica es tradueix directament en un fenomen d'insolubilitat del pèptid durant la síntesi. La necessitat de solubilitzar seqüències quan aquestes es troben protegides, ha requerit el desenvolupament de noves tàctiques per solubilitzar- les i poder aconseguir la seva síntesi, especialment quan mètodes convencionals no serveixen. Donat aquest factor, en els darrers anys s'han proposat diverses estratègies

210 CAPÍTOL 2: Un nou Grup Protector per a Amides de Cadena Peptídica Resum

dirigides a disminuir aquesta associació de ponts d'hidrogen inter- o intramoleculars que afecta la solubilitat dels "pèptids difícils".19–21

Els factors que participen en solucionar la insolubilitat d'aquestes seqüències estan dirigits a solubilitzar pèptids protegits durant la seva síntesi o a solubilitzar-los desprotegits quan es troben en solució. Els dos tipus d'insolubilitats comparteixen el mateix grau d'importància, ja que el primer provoca la formació d'impureses i el segon impossibilita l'anàlisi i purificació. Degut a l'estudi realitzat en aquest capítol, ens centrarem en les estratègies dirigides a solubilitzar seqüències durant la seva síntesi per tal d'obtenir "pèptids difícils". Així doncs, els paràmetres que participen en la solució d'aquest inconvenient poden ser externs si s'afegeixen canvis del dissolvent, salts caotròpiques o detergents;22 o poden referir-se a modificacions químiques quan es manipula internament l'estructura del pèptid. Pel què fa a les alteracions efectuades a la cadena peptídica, les més conegudes es classifiquen en: (i) síntesi d'un anàleg temporal de la seqüència que inclou un enllaç éster que posteriorment es transforma en amida;23 i (ii) la incorporació d'un protector temporal per a l'enllaç amida de la cadena peptídica.24,25 En aquest capítol ens hem centrat en la darrera estratègia per a solubilitzar "pèptids difícils", la protecció d'amida de cadena peptídica.

L'efecte d'incorporació d'un grup en el nitrogen amida evita la presència del protó amida i provoca a la seqüència una interrupció dels continuats ponts d'hidrogen, fent desestabilitzar l'estructura de làmina-β. Quan la molècula s'uneix a l'amida de manera temporal es parla de grup protector, així es pot dir que es protegeix un grup funcional que normalment es troba lliure en la síntesi de pèptids. Cal tenir en compte que aquests protectors, que generalment estan dissenyats per ser eliminats durant la desprotecció global del pèptid, només aporten solubilitat durant la síntesi, ja que quan la seqüència es troba desprotegida ells ja no hi són presents.

Des del 1992 que es van dissenyar les pseudoprolines26 com a grups protectors per a amida, a la literatura se n'han descrit altres, tots encaminats a facilitar la síntesi de "pèptids difícils".27–29 Els protectors d'enllaç amida per a pèptids proposats fins al moment presenten certes limitacions associades a una baixa reactivitat d'incorporació d'aquests; o a un elevat cost econòmic associat a la seva preparació; o a la necessitat que certs AAs siguin presents a la seqüència per tal d'introduir aquest protector.30

Dins del marc de la recerca associada a aquests protectors, i amb la intenció de millorar les limitacions mencionades, s'ha proposat, en el següent capítol, el desenvolupament de la síntesi d'un nou grup per l'amida de cadena peptídica que permeti la síntesi de "pèptids difícils" mitjançant la millora en la seva solubilitat.

211 CAPÍTOL 2: Un nou Grup Protector per a Amides de Cadena Peptídica Resum

Per tal d'assolir aquest objectiu es va proposar la molècula 2-metoxi-4- metilsulfinilbenzil (Mmsb) com a nou grup protector de l'esquelet peptídic. Aquesta molècula havia estat prèviament utilitzada, però en forma de carbamat (Mmsz)31 per a la protecció d'amines i com a espaiador bifuncional. La peculiaritat de la molècula proposada és que es classifica dins del concepte de "safety-catch", que va ser definit el 1971 en base a aquells enllaços que després d'una modificació química, en un moment determinat, poden convertir-se en làbils a certes condicions.32 Aquest comportament va permetre pensar en el disseny d'un grup protector de tipus semi-permanent: estable al medi bàsic utilitzat durant la síntesi del pèptid per 9-fluorenilmetoxicarbonil (Fmoc)/tert-butil (tBu) en SPPS, i alhora estable al medi àcid associat a l'escissió del pèptid, però que després d'un tractament químic el grup es tornaria làbil a àcid (Fig. 4).

En tant que grup protector d'amida de cadena peptídica, l'Mmsb pretén facilitar la solubilitat de la seqüència en fase sòlida (durant la síntesi), permetent una incorporació millorada dels AAs. Però alhora, aquest grup aporta una nova avantatge que no la presenten la gran majoria dels grups protectors d'amida: la possibilitat d'obtenir la seqüència semi-protegida després de fer l'escissió del pèptid de la resina, que fa millorar la seva solubilitat en solució. Aquesta última qualitat evita l'agregació del pèptid en solució, facilitant-ne la caracterització i la seva purificació, etapa que amb l'absència d'aquest grup no hauria estat possible. Posteriorment a la escissió del pèptid, mitjançant un tractament reductiu del sulfòxid per donar un tioéter, el grup perd el caràcter electro-atractor fent que en medi àcid es torni làbil i s'afavoreixi la seva eliminació, alliberant-se la seqüència desitjada.

Boc O Desprotecció Global O N N R TFA 95% R tBu Pbf

Metoxi ortho Sulfòxid para Intermedi més soluble aporta labilitat als àcids aporta estabilitat als àcids i (durant la reducció acidolítica) evita agregacions Facilita la Purificació

Reducció acidolítica (NH4I-TFA) O N

R

Figura 4. Esquema general de la metodologia d'utilització del grup protector d'esquelet peptídic Mmsb.

Per tal de dur a terme la incorporació de l'Mmsb a la seqüència peptídica es va decidir introduir-lo a un AA quiral representatiu, com va ser l’alanina (Fmoc-N(Mmsb)-L-Ala-

212 CAPÍTOL 2: Un nou Grup Protector per a Amides de Cadena Peptídica Resum

OH). La síntesi en solució d'aquest AA modificat es va basar en la preparació del Mmsz descrita per Thennarasu i col·laboradors,31 amb certes modificacions per tal d'optimitzar-ne la puresa (Fig. 5). De manera que en cinc etapes i una única purificació (segona reacció per separar el compost 3 del seu regioisòmer formilat a la posició 5) es va obtenir el Fmoc-N(Mmsb)-L-Ala-OH amb una puresa del 98% i un rendiment, no optimitzat, del 19%. Sense cap més purificació aquest producte es va utilitzar directament en les estratègies de totes les síntesis de pèptids modificats amb l'Mmsb mitjançant fase sòlida Fmoc/tBu.

MeI (1.1 eq) DMF-POCl3 (6 eq) Et3N (1.1 eq) CH2Cl2 anh. 0 C 50 C, 15 h 1 CH2Cl2, rt, 30 min 2 3 H-L-Ala-OH (0.4 eq) NaBH3CN (1.4 eq) Dioxà-H2O (1:1) rt, O.N

Fmoc-OSu H2O2 30% (1:100 w/v) (0.9 eq) AcOH (1:400 w/v) Dioxà-H2O (1:1) pH 8

6 5 4

Figura 5. Procés sintètic per a la obtenció de Fmoc-N(Mmsb)-L-Ala-OH.

Per tal d'avaluar l’estratègia esmentada es va introduir l'Fmoc-N(Mmsb)-L-Ala-OH a tres models de pèptids coneguts com a "pèptids difícils": H-(Ala)10-NH2 (decaalanina, utilitzat a la bibliografia com a model de seqüència problemàtica),33,34 Ac-(Arg-Ala-Asp- 13 Ala)4-NH2 (RADA-16), i la Aβ(1-42) (H-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL- MVGGVVIA-OH, pèptid de 42 AAs conegut per la seva implicació en la malaltia de l'alzheimer).35 La síntesi d'aquests pèptids es va fer en fase sòlida i amb la incorporació de l'alanina protegida amb l'Mmsb en una única posició del pèptid: per a la decaalanina es van fer dos anàlegs amb introducció al tercer i cinquè AA, respectivament; per al RADA-16 en el cinquè AA; i finalment per la Aβ(1-42) en el vint- i-dosè AA. Tot i la confirmació de la completa incorporació dels AAs mitjançant el test qualitatiu Kaiser, les síntesis van presentar una dificultat comuna causada a l'impediment estèric del Mmsb, en el punt d'incorporació de l'AA consecutiu a l'Fmoc- N(Mmsb)-L-Ala-OH. Es va realitzar aquesta incorporació en diferents condicions i es van trobar que els millors resultats s'obtenien després de tres incorporacions consecutives de 2 h amb els agents d'acoblament N,N’-diisopropilcarbodiimida (DIPCDI) i 2-ciano-2-hidroxiiminoacetat d'etil (OxymaPure) en MeCN/DMF (3:1) a T= 45 ºC i augmentant els equivalents d'AA i agents d'acoblament.

213 CAPÍTOL 2: Un nou Grup Protector per a Amides de Cadena Peptídica Resum

Una alternativa proposada per tal de facilitar la incorporació de l'AA consecutiu a l'AA sintetitzat protegit amb l'Mmsb, va consistir en la síntesi en solució del dipèptid Fmoc- L-Ala-N(Mmsb)-L-Ala-OH per a la decaalanina. La formació de l'enllaç amida es va realitzar amb la prèvia activació de l'àcid carboxílic de l'alanina comercial com a fluorur d'àcid (Fmoc-L-Ala-F), per després fer-lo reaccionar amb el corresponent grup amino de l'N(Mmsb)-L-Ala-OH. L'acoblament del dipèptid per a fer la decaalanina en fase sòlida es va poder obtenir completament en condicions estàndards utilitzant PyBOP i DIEA. Després de l'escissió del pèptid de la resina es va poder confirmar la validesa del mètode sense incorporacions incompletes d'AA, amb la obtenció de la decaalanina amb una puresa molt elevada.

Un cop complerta l’elongació dels pèptids amb l'Mmsb, aquests es van escindir de la resina amb TFA 95% [combinat amb triisopropilsilà (TIS) 2.5%, i H2O 2.5% quan els pèptids contenien cadenes laterals d'AA protegides], corroborant-ne per cromatografia i espectrometria de masses que tots ells contenien el grup Mmsb i la resta d'AAs desprotegits. Mitjançant anàlisis d'HPLC i espectrometria de masses es va confirmar que a nivell sintètic la incorporació de l'Mmsb a la seqüència produïa productes amb menys impureses que les síntesis que no tenien el grup protector, els quals majoritàriament contenien delecions de certs AAs. Per altra banda, l'anàlisi de solubilitat va permetre confirmar que els pèptids semi-protegits amb l'Mmsb eren notablement més solubles que els seus homòlegs totalment desprotegits.

Amb la decaalanina es van realitzar anàlisis amb dicroisme circular tant de la seqüència sense grup protector, com dels dos anàlegs protegits amb l'Mmsb, observant-se clarament que el pèptid desprotegit presentava una estructura secundària de làmina-β, mentre que els dos protegits mostraven una estructura desordenada "random coil". Aquesta diferència estructural pot entendre's com una demostració de l'efecte causat per la presència de l'Mmsb en el plegament del pèptid en solució, i per tant un motiu explicatiu de l'augment de solubilitat i la obstrucció en l'autoensamblatge.

Amb els altres dos pèptids modificats amb l'Mmsb, el RADA-16 i la Aβ(1-42), es van realitzar les seves purificacions per HPLC semi-preparatiu, i gracies a l'augment de la solubilitat associada al protector, els dos pèptids van poder ser aïllats amb una elevada puresa.

Finalment, l'etapa de desprotecció per a obtenir els pèptids diana va consistir en l'eliminació del grup Mmsb mitjançant la reducció acidolítica amb NH4I en TFA, succeint dues reaccions alhora, la reducció i la desprotecció. Seguint la reacció per espectrometria de masses es va poder observar que en presència de NH4I, el sulfòxid

214 CAPÍTOL 3: Estratègies de Conjugació amb Pèptids Solubilitzadors Resum

de l'Mmsb es reduïa i es tornava làbil a àcid provocant la immediata alliberació del pèptid desprotegit.

En aquest capítol s'ha pogut concloure que la incorporació del nou grup protector per l'amida de l'esquelet peptídic, Mmsb, ha contribuït a facilitar la síntesi de "pèptids difícils" en fase sòlida, alhora que ha permès la seva manipulació en solució. La síntesi del grup protector s'ha realitzat mitjançant la seva inserció en el grup α-amino de l'Ala [Fmoc-N(Mmsb)-L-Ala-OH]. La seva incorporació s'ha dut a terme en condicions estàndards en fase sòlida en una posició de la seqüència racionalment seleccionada per als pèptids models: la decaalanina, el RADA-16 i la Aβ(1-42).

La peculiaritat de l'Mmsb, en tant que grup tipus "safety-catch", s'ha demostrat pel què fa a la seva estabilitat al medi bàsic durant la síntesi i al medi àcid durant l’escissió del pèptid de la resina. La millora en les síntesis en fase sòlida associades a la presència de l'Mmsb, comparat amb les que no el contenien, s'ha corroborat fent-se evident en tant que ha evitat la deleció d'AAs. La implicació de l'Mmsb en evitar associacions tipus làmina-β en solució s'ha demostrat per dicroisme circular per a la decaalanina. La solubilitat dels pèptids semi-protegits amb l'Mmsb ha estat notablement augmentada comparada amb els pèptids sense protegir, fet que ha permès la seva caracterització per HPLC, així com la purificació del RADA-16 i la Aβ(1-42). L'eliminació del grup protector "safety-catch" Mmsb s'ha dut a terme mitjançant una reducció acidolítica obtenint tots els exemples de "pèptids difícils" desprotegits mantenint-ne la puresa de síntesi.

Capítol 3: Estratègies de Conjugació amb Pèptids Solubilitzadors

En el marc de la investigació associada a la millora de la solubilitat dels pèptids, aquesta vegada s'ha dirigit cap a les seqüències obtingudes després de la desprotecció global, en solució. Donada la importància, comentada anteriorment, d'obtenir mostres solubles per a possibilitar la seva caracterització i purificació, s'han desenvolupat diferents eines sobre aquest camp. Centrant-nos en aquest sector tractat en aquest capítol trobem descrites estratègies basades en factors externs i altres en modificacions químiques. Pel què fa als primers trobem: la selecció de dissolvents, el pH, l'addició de sals caotròpiques, i l'addició de detergents.38 En canvi pel què fa a les alteracions efectuades a la cadena peptídica, les més conegudes es classifiquen en: (i) incorporacions temporals o permanents de molècules polars com el polietilè glicol (PEG)39 o els sucres;40 (ii) conjugacions temporals o permanents de segments peptídics

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curts i polars.41 En aquest capítol concretament s'estudiarà aquesta última estratègia basada en la conjugació de pèptids de cadena curta amb capacitat solubilitzadora. Aquesta eina es pot aplicar tant a molècules orgàniques petites com a cadenes aminoacídiques.

Les seqüències curtes de pèptids utilitzades per a aquesta finalitat són homooligopèptids composats per AAs de cadena lateral carregada positiva- (Arg o Lys) o negativament (Asp o Glu).36,37 De manera que la unió d'un d'aquests pèptids a una molècula/pèptid insoluble en aigua, aporta un component iònic que li fa augmentar la solubilitat i que pot estar connectat a la molècula d'estudi de manera temporal o permanent. Un dels avantatges per les quals aquesta estratègia suposa una alternativa a altres mètodes rau en la facilitat de conjugació sintètica, que s'aconsegueix mitjançant la síntesi en fase sòlida.

A la literatura es troba àmpliament estudiada aquesta estratègia per a proteïnes,41 però menys aplicada als pèptids. S'ha descrit l'ús d'homooligopèptids que es connecten a proteïnes amb la finalitat d'aportar solubilitat, per facilitar sobretot la purificació d'aquestes.42 Pel què fa als pèptids apolars amb conjugació permanent a aquestes seqüències curtes carregades, s'han descrit casos en combinació amb PEG43 on s'aconsegueixen formació de micel·les o fins i tot dendrímers.44 Aquestes estructures s'han utilitzat principalment com a sistemes d'alliberament de fàrmacs i han aprofitat l'avantatge d'AAs com la Lys que té un alt grau de ramificació.45 La forma globular que presenten donen alhora un augment de punts d'enllaç per a les molècules apolars d'estudi, característica aprofitada en tractaments de transport de fàrmacs.

Per altra banda, quan el pèptid insoluble i el pèptid curt solubilitzador s'uneixen mitjançant una conjugació temporal, és necessari la incorporació d'un espaiador bifuncional entre la seqüència d'estudi i el pèptid solubilitzador. La síntesi d'aquest conjugats també es realitza íntegrament en fase sòlida, i un cop escindit el pèptid de la resina, la seqüència apolar s'obté connectada a través de l'espaiador bifuncional al pèptid solubilitzador. L'estabilitat del connector a les condicions d'escissió és imprescindible, permetent que la seqüència es pugui manipular en solució, ja que així n'haurà augmentat la seva solubilitat en aigua. Posteriorment, un tractament específic facilita la desconnexió entre pèptid apolar i l'espaiador junt amb el pèptid solubilitzador, alliberant-se així el pèptid desitjat.

A la literatura es troben pocs casos enfocats a solubilitzar pèptids amb l'estratègia de connexió temporal. Durant els darrers anys han destacat treballs on solubilitzadors del

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46 47 48 49,50 tipus (Gly-Arg)4, (Gly-Arg/Lys)2-6, o homololigopèptids d'Arg o de Lys han estat connectats a través d'espaiadors làbils a àcid50 o base.46,48,49

En el següent capítol, en base a l'estratègia de solubilitzar molècules/pèptids apolars amb la unió d'homooligopèptids s'han realitzat diferents estudis, (i) inicialment, comparar les diferències de polaritat aportades segons el tipus d'AA i la seva disposició a l'espai d'estructures peptídiques solubilitzadores. Per altra banda, (ii) avaluar l'eficàcia de l'estructura Mmsb-OH com a espaiador bifuncional per connectar un pèptid insoluble a una cadena curta solubilitzadora, de manera temporal. Com a darrer objectiu, de manera paral·lela, (iii) s'ha decidit ampliar l'aplicació de l'espaiador bifuncional Mmsb-OH com a grup protector C-terminal per a permetre la síntesi de pèptids mitjançant la condensació de fragments en solució.

El dipèptid di-naftilalanina [H-(Nal)2-NH2], va servir de model apolar per al disseny de conjugació permanent a un conjunt de cadenes curtes solubilitzadores que tinguessin 4 grups polars lliures (2, 3, 4 i 8, Fig. 6), o bé amino (aportats per les Lys) o bé guanidini (aportats per les Arg), combinant pèptids lineals o ramificats. La síntesi d'aquests pèptids units al model apolar es va dur a terme totalment en fase sòlida seguint l’estratègia Fmoc/tBu. L’escissió dels pèptids de la resina es va fer amb TFA 95% per tal d'eliminar els grups protectors tant de les Lys com de les Arg. El posterior anàlisi cromatogràfic per HPLC va permetre avaluar la polaritat de cadascun d'ells.

1 H-(Nal)2-NH2 2 H-(Lys)3-(Nal)2-NH2 3 H-(Arg)3-(Nal)2-NH2 4 Ac-(Arg)4-(Nal)2-NH2

5 H-(Lys)7-(Nal)2-NH2 6 H-(Arg)7-(Nal)2-NH2 7 Ac-(Arg)8-(Nal)2-NH2 Pèptids Lineals Pèptids Ramificats

8 9

Figura 6. Estructura química dels pèptids sintetitzats.

La primera conclusió que es va extreure d'aquest experiment va ser que els grups amino aportaven una polaritat superior als grups guanidini; i per altra banda s'intuïa que el pèptid ramificat encara donava més polaritat que el seu homòleg lineal. Per tal de confirmar la tendència intuïda amb els primers pèptids, es va voler fer un pas més

217 CAPÍTOL 3: Estratègies de Conjugació amb Pèptids Solubilitzadors Resum

ampliant el nombre de grups polars fins a 8, de manera que es va preparar una segona generació de pèptids (5, 6, 7 i 9, Fig. 6).

Un cop es van escindir els pèptids de la resina es van analitzar per cromatografia i es va observar que el poder per incrementar la polaritat de la molècula model s'accentuava més com més grups polars hi havia. De manera que la segona generació va permetre confirmar el què ja s'apuntava a la primera generació de pèptids, on el pèptid ramificat 9 era, amb diferència, el que més polaritat donava.

Un últim disseny peptídic (10, Fig. 7) va permetre comprovar la importància en termes de polaritat de la disposició espaial dels grups polars. Així que es va realitzar la síntesi d'un nou pèptid de 8 lisines disposades de manera que exposessin 4 grups amino de tipus α i 4 de tipus ε (comparable amb el 5 que en té 1 α i 7 ε i amb 9 que en té 4 α i 4 ε). De manera que el 10 compartia semblances amb el 9 pel què fa a la naturalesa dels grups amino exposats al medi, i semblances amb el 5 pel què fa a la disposició espaial lineal. L'anàlisi per HPLC va resoldre el dubte portant a la conclusió que, entre els tres, el ramificat 9 seguia sent el que aportava més polaritat, i que el 10 tenia una polaritat semblant a 5. Es demostrava la importància de la disposició a l'espai dels grups polars, més que no pas la naturalesa d'aquests grups.

Per tal de tenir donar una aplicació utilitzant molècula coneguda es va decidir agafar el pèptid que millors resultats de polaritat havia donat (9) i unir-lo a un fàrmac comercial insoluble en aigua (Indometacina, 11, Fig. 7). La síntesi es va fer íntegrament en fase sòlida, unint el fàrmac a través de la cadena lateral de la primera lisina introduïda com Fmoc-Lys(Mtt)-OH, després de l’eliminació del 4-metiltritil (Mtt). L’escissió del pèptid conjugat al fàrmac de la resina i posterior purificació va permetre la comparativa cromatogràfica que demostrava el notable increment de la polaritat del fàrmac. De la mateixa manera, es va analitzar la solubilitat en aigua a la mateixa concentració del fàrmac sol i d'aquest, conjugat, i es va observar clarament la millora de solubilitat quan aquest estava conjugat a les lisines ramificades.

218 CAPÍTOL 3: Estratègies de Conjugació amb Pèptids Solubilitzadors Resum

11

10

Figura 7. Estructura química de: (10) el pèptid amb 8 Lys i 4 grups amino tipus α i 4 tipus β; i (11) la Indometacina.

En relació al segon objectiu, aprofitant els avantatges obtinguts al capítol segon amb l'estructura Mmsb, en aquest capítol es van estudiar els beneficis d'utilitzar-lo com a espaiador bifuncional. La característica principal que l'Mmsb-OH comparteix amb el grup protector d'amida de cadena peptídica estudiat al capítol segon és relativa a la seva estructura, que el fa estable a condicions acídiques elevades, però làbil un cop reduït el sulfòxid. L'estudi realitzat amb aquesta molècula es va fer amb la química de pèptids en fase sòlida Fmoc/tBu, aportant una primera novetat, ja que a la literatura es descriu l'ús de l'Mmsb-OH només en la química tert-butiloxicarbonil (Boc)/benzil (Bzl). La síntesi d'aquest espaiador bifuncional es va dur a terme seguint la mateixa ruta sintètica descrita per Thennarasu i col·laboradors, optimitzant-ne aquells passos que donaven rendiments baixos o que obligaven a fer una purificació.

Per tal de demostrar els avantatges d'introduir aquest espaiador com a grup protector semi-permanent es va escollir un pèptid conegut (Q11, Fig. 8) amb una seqüència amb característiques d'insolubilitat en medi aquós. Entre aquest espaiador bifuncional i la resina es va decidir introduir un conjunt de sis lisines com a pèptid solubilitzador, pretenent connectar un element de grups polars que aportés solubilitat (poly-Lys) a un pèptid clarament insoluble (Fig. 8). D'aquest mode, temporalment es té una seqüència soluble fàcilment tractable en solució, caracteritzable i purificable que permet, mitjançant un últim pas, separar aquesta unió i tenir el pèptid objectiu (Q11).

219 CAPÍTOL 3: Estratègies de Conjugació amb Pèptids Solubilitzadors Resum

pèptid Q11 espaiador (Lys)6 bifuncional Mmsb-OH

Figura 8. Seqüència del pèptid Q11 unit a través de l'espaiador bifuncional Mmsb a la poly-Lys.

La síntesi d'aquesta molècula es va fer totalment en fase sòlida i l’escissió d'aquesta de la resina es va dur a terme en condicions fortament àcides TFA/TIS/H2O (95:2.5:2.5), de manera que es va obtenir l’estructura de la Figura 8. Posteriorment es va realitzar un seguiment amb HPLC i MALDI-TOF per tal de corroborar que, amb les mateixes condicions utilitzades per eliminar el grup Mmsb (NH4I/TFA), es podia separar la seqüència Q11 del conjunt de lisines, quedant així el pèptid desitjat lliure.

Pel què fa al darrer objectiu, relatiu a una altra aplicació del mateix espaiador bifuncional, l'Mmsb-OH es va avaluar la possibilitat que formés part d'una seqüència com a grup protector C-terminal. Donada la necessitat de grups protectors d'àcid carboxílic en les estratègies de condensació de fragments en solució quan es volen obtenir pèptids àcid carboxílic en el seu extrem C-terminal, es va voler contribuir en aquest camp.51 L'avaluació com a tal es va fer amb la condensació de dos fragments per a obtenir un segment de la seqüència d'un pèptid conegut de propietats terapèutiques per a la diabetis, l'Exenatide (NH2-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly- Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH). Aprofitant la prèvia síntesi de l'Mmsb-OH, i la seva posterior introducció en fase sòlida, es va preparar el fragment C-terminal sense complicacions i amb una puresa elevada. Paral·lelament, també en fase sòlida es va sintetitzar el segon fragment, l'N-terminal, que contenia el grup Fmoc en el seu extrem amino de la cadena. Els dos pèptids van ser escindits de la resina independentment en condicions de TFA 1% que permetien la obtenció d'aquests amb les cadenes laterals protegides, així com la presència de l'Mmsb al fragment C-terminal.

La condensació de fragments en solució es va dur a terme amb l'agent d'acoblament PyBOP en presència de base, aconseguint el fragment desitjat amb una puresa acceptable del 92% per HPLC. La posterior eliminació del grup Fmoc en l'amino de l'N- terminal es va realitzar mantenint-se la puresa de l’etapa anterior. Finalment, l'eliminació del grup protector d'àcid carboxílic de l'extrem C-terminal mitjançant les condicions de reducció acidolítiques descrites pel grup protector d'amida Mmsb, van

220 CAPÍTOL 3: Estratègies de Conjugació amb Pèptids Solubilitzadors Resum

permetre aïllar-ne el segment d'Exenatide desitjat sense haver augmentat el grau d'impureses.

En aquest capítol, en base als objectius plantejats amb les estructures poly-Lys i poly- Arg com a pèptids solubilitzadors units permanentment a un model de pèptid apolar, les polaritats obtingudes per HPLC van permetre deduir que: i) la seqüència poly-Lys lineal aporta més polaritat que la poly-Arg lineal; ii) l'estructura ramificada de poly-Lys contribueix en major grau a l'augment de la polaritat que la seva homòloga lineal; iii) el tipus de grup amino (α or ε) exposat al medi aquós influencia menys en la polaritat que no pas la distribució espaial d'aquest grups. Finalment, l'exemple de la seqüència solubilitzadora més polar de les proposades es va conjugar a un fàrmac apolar validant així l'eficàcia de l'augment de polaritat i solubilitat atribuït a aquesta estratègia.

Pel què fa a la mateixa estratègia de conjugació a una seqüència curta de pèptid solubilitzador es va dissenyar amb l'ús de l'espaiador bifuncional Mmsb-OH una unió temporal per a pèptids insolubles. L'estabilitat i labilitat posterior de l'espaiador bifuncional es va demostrar durant la síntesi, escissió del pèptid i trencament de l'enllaç d'unió amb la seqüència solubilitzadora. La cadena curta de 6 Lys va ser l'escollida per a facilitar l'anàlisi d'un pèptid insoluble en aigua, i el seu augment de solubilitat va corroborar l'objectiu assolit amb aquesta l'estratègia.

Com a darrer punt, pel mateix espaiador bifuncional Mmsb-OH s'ha ampliat el rang d'aplicacions, aquesta vegada emprat com a protector d'àcid carboxílic per al C•terminal. La condensació de dos fragments en solució d'un segment d'un pèptid conegut en va confirmar la seva validesa. L'escissió dels fragments mantenint les cadenes laterals dels AAs protegides i l'estabilitat també del protector Mmsb van permetre unir-los en solució amb una puresa adequada. Per finalment obtenir el segment desitjat es van eliminar alhora els protectors de les cadenes laterals d'AAs, així com el protector C-terminal, mitjançant una reducció acidolítica, fet que va validar la proposta d'aquest nou grup protector per a àcids carboxílics.

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